12124391
full text
S67K
protein
substitution
true negative
Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K currents.
A depolarizing pre-pulse relieved G tion of Ca2 currents by the wild type and the S98T and T128F mutants but not the S67K mutant.
Both wild type subunits activated phospholipase C 2 and mutant G with similar potencies; however, the S67K mutant showed reduced maximal activity.
One of these mutants, G 1(S67K) 2 showed specific functional defects in regulating each of these three effectors without affecting other G functional interactions with these effectors.
GST-GIRK Pulldown Assays--Recombinant baculoviruses encoding wild type 1, 1(S67K), 1(S98T), and 1(T128F) were generated using the Bac to Bac Expression System (Invitrogen).
G Mutants Confer Functional but Not Binding Defects--We previously reported that the G 1 S67K, T128F, and S98T mutants failed to activate heteromeric GIRK1/GIRK4 channels.
Wild type G 1 as well as mutants G 1(S67K), G 1(S98T), and G 1(T128F), all bound both the N and C termini of GIRK4.
Although some differences in channel binding could be detected between G 1(S67K) and wild type G 1, precise quantitation of such differences was not attempted.
The G 1(S67K) mutant on the other hand failed to activate the channel in half of the patches tested, whereas the activation in the other half of the patches was minor (Fig.
However, in all patches regardless of the ability of S67K to activate GIRK* currents, the Na response was enhanced following application of S67K (Fig.
Point mutations (S67K, S98T, and T128F) and wild type G 1 bind the GIRK4 C- and N-terminal domains but fail to activate the channel.
Mutants G 1(S67K), G 1(S98T), and G 1(T128F) showed significantly reduced enhancement of GIRK4* basal current as compared with the wild type G 1 (*, p 0.01; unpaired t test, n 5 6).
These results indicate that S67K induces the conformational changes on the channel that are necessary to enhance the Na activation but are not sufficient to cause channel activation.
Furthermore, in those patches where channel activity was stimulated by S67K, this activity was smaller (Fig.
We tested the interactions of wild type G 1 2 as well as the three mutants S67K, S98T, and T128F with N-type channels expressed in Xenopus oocytes.
Mutant S67K did not activate the channel in three patches of six tested, and in those where it did activate, it could be washed almost completely within 30 s.
S67K activated channels in half of the patches tested.
In oocytes expressing G 1(S67K) 2 basal current inhibition was similar to other mutants ( 50%, Fig.
We tested the effectiveness of our three mutants, S67K, S98T, and T128F in activating PLC 2.
Interestingly, G 1(S67K) 2 showed reduced maximal activation of PLC 2, suggesting partial activation of the enzyme and a potential role for this residue in functional interactions between the two proteins.
Pre-pulse depolarization relieves inhibition by mutants S98T and T128F but not S67K.
Wild type G 1 2, S98T, and T128F all mediated pre-pulse facilitation, whereas S67K did not (*, significantly different from G 1 2; p 0.01; unpaired t test, n 8).
Interestingly, pre-pulse depolarization did not relieve the inhibition by the S67K mutant, whereas the other two mutants behaved li wke wild type G .
The mutant S67K is immune to the voltage-dependent relief although it effectively interacts and inhibits the channel.
However, S67K showed reduced maximal activity.
The G 1 mutant S67K activates phospholipase C 2 at submaximal levels.
All tested combinations enhance basal PLC activity with similar potencies (EC50 values in nM: G 1 2 20.8; S67K 22.0; S98T 17.6; T128F 26.7).
The maximal effectiveness of S67K is reduced compared with the wild type G 1.
S143T
protein
substitution
true negative
Oocytes were injected with cRNAs, 2 ng each of GIRK4(S143T) and 2 ng of each G protein subunit or mutant.
Here, we use GIRK4(S143T) (denoted as GIRK4*), a homomeric-active GIRK4 channel with a mutation in the poremutants to helix (11), to test the effectiveness of these G enhance basal currents.
T128F
protein
substitution
true negative
Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K currents.
A depolarizing pre-pulse relieved G tion of Ca2 currents by the wild type and the S98T and T128F mutants but not the S67K mutant.
GST-GIRK Pulldown Assays--Recombinant baculoviruses encoding wild type 1, 1(S67K), 1(S98T), and 1(T128F) were generated using the Bac to Bac Expression System (Invitrogen).
G Mutants Confer Functional but Not Binding Defects--We previously reported that the G 1 S67K, T128F, and S98T mutants failed to activate heteromeric GIRK1/GIRK4 channels.
Wild type G 1 as well as mutants G 1(S67K), G 1(S98T), and G 1(T128F), all bound both the N and C termini of GIRK4.
Mutants S98T and T128F also activated the channel in all patches that were tested; however, the activation was smaller, and mutants could be washed out with faster kinetics compared with the wild type G (Fig.
Point mutations (S67K, S98T, and T128F) and wild type G 1 bind the GIRK4 C- and N-terminal domains but fail to activate the channel.
Mutants G 1(S67K), G 1(S98T), and G 1(T128F) showed significantly reduced enhancement of GIRK4* basal current as compared with the wild type G 1 (*, p 0.01; unpaired t test, n 5 6).
We tested the interactions of wild type G 1 2 as well as the three mutants S67K, S98T, and T128F with N-type channels expressed in Xenopus oocytes.
Most of the activity of mutants S98T and T128F were washed with faster kinetics (less than 30 s).
S98T and T128F activated channels in all patches tested.
Pre-pulse facilitation was observed in oocytes expressing G 1 2, G 1(S98T) 2 and G 1(T128F) 2.
Both S98T and T128F mutants showed facilitation that was not significantly different from the wild type G 1 2 (p 0.05, unpaired t test).
We tested the effectiveness of our three mutants, S67K, S98T, and T128F in activating PLC 2.
Pre-pulse depolarization relieves inhibition by mutants S98T and T128F but not S67K.
Wild type G 1 2, S98T, and T128F all mediated pre-pulse facilitation, whereas S67K did not (*, significantly different from G 1 2; p 0.01; unpaired t test, n 8).
All tested combinations enhance basal PLC activity with similar potencies (EC50 values in nM: G 1 2 20.8; S67K 22.0; S98T 17.6; T128F 26.7).
S98T
protein
substitution
true negative
Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K currents.
A depolarizing pre-pulse relieved G tion of Ca2 currents by the wild type and the S98T and T128F mutants but not the S67K mutant.
GST-GIRK Pulldown Assays--Recombinant baculoviruses encoding wild type 1, 1(S67K), 1(S98T), and 1(T128F) were generated using the Bac to Bac Expression System (Invitrogen).
G Mutants Confer Functional but Not Binding Defects--We previously reported that the G 1 S67K, T128F, and S98T mutants failed to activate heteromeric GIRK1/GIRK4 channels.
Wild type G 1 as well as mutants G 1(S67K), G 1(S98T), and G 1(T128F), all bound both the N and C termini of GIRK4.
Mutants S98T and T128F also activated the channel in all patches that were tested; however, the activation was smaller, and mutants could be washed out with faster kinetics compared with the wild type G (Fig.
Point mutations (S67K, S98T, and T128F) and wild type G 1 bind the GIRK4 C- and N-terminal domains but fail to activate the channel.
Mutants G 1(S67K), G 1(S98T), and G 1(T128F) showed significantly reduced enhancement of GIRK4* basal current as compared with the wild type G 1 (*, p 0.01; unpaired t test, n 5 6).
We tested the interactions of wild type G 1 2 as well as the three mutants S67K, S98T, and T128F with N-type channels expressed in Xenopus oocytes.
Most of the activity of mutants S98T and T128F were washed with faster kinetics (less than 30 s).
S98T and T128F activated channels in all patches tested.
Pre-pulse facilitation was observed in oocytes expressing G 1 2, G 1(S98T) 2 and G 1(T128F) 2.
Both S98T and T128F mutants showed facilitation that was not significantly different from the wild type G 1 2 (p 0.05, unpaired t test).
We tested the effectiveness of our three mutants, S67K, S98T, and T128F in activating PLC 2.
Pre-pulse depolarization relieves inhibition by mutants S98T and T128F but not S67K.
Wild type G 1 2, S98T, and T128F all mediated pre-pulse facilitation, whereas S67K did not (*, significantly different from G 1 2; p 0.01; unpaired t test, n 8).
All tested combinations enhance basal PLC activity with similar potencies (EC50 values in nM: G 1 2 20.8; S67K 22.0; S98T 17.6; T128F 26.7).
12397059
full text
H282D
protein
substitution
true positive
Q61180
The mutation H282D or double mutations H282R/ H239R eliminated Ni2 block.
4A, all four mutations ( H282C, H282R, H282W, and H282D) dramatically ded creased Ni2 inhibition as evidenced by the shift in the Ni2 ose-response curves of the mutant channels to the right with respect to that of WT.
The changes in Ni2 inhibition observed H282W wH h these mutant channels were H282D it 282R H282C.
No inhibition of amiloridesensitive Na currents by Ni2 was observed in oocytes exi pressing H282D- - mENaCs.
On the contrary, external Ni2 nduced a significant increase in H282D- - currents at concentrations of 0.01, 0.1, and 1 mM, with peak stimulation at 0.1 mM.
For H282D- - mENaC, current rectification was evident in the absence of Ni2 (Fig.
A, dose-response c ( urves of external Ni2 on H282R- ), H282D- - ( ), H282C- - (OE), and H282W- - (q) mENaCs.
Numbers of clamped oocytes are 4 for H282C- a nd H282W- - , 5 for H282R- - , and 14 for H282D- - .
Moreover, the observed Ni2 inhibition of ENaC currents was specifically eliminated by H282D mutation and by the double ( H282R/ H239R) mutation, which strongly suggests that the whole-cell current reduction is due to Ni2 interaction with ENaC rather than with other channels.
The largest change in Ni2 dose response on mENaC currents was observed with H282D (Fig.
Ni2 dose-response curves for other mutant channels (except H282D) showed similar shifts in both the high and low affinity binding sites.
The elimination of Ni2 nhibition by point mutation H282D, the enhanced Ni2 inhibition by R280H, and partial reversal of the enhancement by a mutation in ENaC ( H239R) also support this view.
The observations that H282D or H282R/ H239R eliminated both high and low affinity inhibition are not consistent with the possibility.
Although we do not have direct evidence for the involvement of two His282 residues in Ni2 coordination, the loss of Ni2 block observed with H282D- - channels, compared with the modest change in Ni2 sensitivity observed with - - H239D channels, is consistent with the notion that more than one -subunit participates in the coordinated binding of Ni2 .
N285H
protein
substitution
true positive
Q61180
In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/ H282 (referred to as His279 282) and H282/Y283H/I284H/ N285H (referred to as His282285), were also generated.
Three other mutations b ( W278H, Y283H, and N285H) slightly decreased Ni2 lock of the Na currents, whereas three other mutations ( Y279H, F281H, and I284H) did not significantly change Ni2 block (Fig.
His279 282 and His282285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H.
Q220H
protein
substitution
Q9WU38
true positive
Channel n Ki mM Hill coefficient R2 -H381R- - H319R- - H338R - Q220H- 6 6 8 6 6 0.58 0.73 0.82 0.97 1.03 0.09 0.15 0.12 0.22 0.10a 0.37 0.42 0.33 0.39 0.33 0.01 0.02 0.02 0.03 0.01 0.9545 0.9891 0.9846 0.9895 0.9529 0.0116 0.0022 0.0025 0.0022 0.0054 a p 0.01 from Student's t tests between WT and the mutant channel.
Introduction of a mENaC histidine residue at the corresponding site in ( Q220H) resulted in a modest increase in Ni2 Ki, suggesting i that this residue does not have an important role in Ni2 nhibition of ENaC (Table I).
F281H
protein
substitution
true positive
Q9WU39
In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/ H282 (referred to as His279 282) and H282/Y283H/I284H/ N285H (referred to as His282285), were also generated.
Three other mutations b ( W278H, Y283H, and N285H) slightly decreased Ni2 lock of the Na currents, whereas three other mutations ( Y279H, F281H, and I284H) did not significantly change Ni2 block (Fig.
His279 282 and His282285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H.
H282C
protein
substitution
true positive
Q61180
Although H282C- - channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), - - H239C channels were insensitive to MTSET.
4A, all four mutations ( H282C, H282R, H282W, and H282D) dramatically ded creased Ni2 inhibition as evidenced by the shift in the Ni2 ose-response curves of the mutant channels to the right with respect to that of WT.
The changes in Ni2 inhibition observed H282W wH h these mutant channels were H282D it 282R H282C.
Only the dose-response data for H282C- mENaCs were fitted reasonably well with the one-site equation that yielded parameters as follows: Ki, 7.46 mM; Hill coefficient, 0.45; and R2, 0.968.
MTSET Inhibited Amiloride-sensitive Na Currents in Oocytes Expressing H282C- - mENaCs--The above results suggested that a Ni2 -binding site consisting of His282 and His239 has a key role in Ni2 inhibition of ENaC currents.
To explore further the role of His282 in channel activity, we examined the response of the mutant channels ( H282C- - ) to external sulfhydryl reagents.
The negatively charged reagent MTSES [sodium (2-sulfonatoethyl) methanethiosulfonate] at 5 mM also inhibited about 40% of the Na currents of H282C- - channels, whereas it did not change the currents of WT channels (24).
MTSET and MTSES blocked H282C .
A, dose-response c ( urves of external Ni2 on H282R- ), H282D- - ( ), H282C- - (OE), and H282W- - (q) mENaCs.
Numbers of clamped oocytes are 4 for H282C- a nd H282W- - , 5 for H282R- - , and 14 for H282D- - .
MTSET reduced Na currents in oocytes expressing H282C- - mENaC and further reduced Ni2 inhibition of the remaining currents.
A, effects of MTSET on H282C- - and - - H239C mENaCs.
Open, filled, and shaded bars are the relative currents (mean S.E., n 5 for H282C- - and n 13 for - - H239C) obtained before and after MTSET and after washout of MTSET, respectively.
C, Ni2 dose responses on H282C- - mENaC currents without (E) and with (q) treatment of the oocytes with 1 mM MTSET.
The dose response of Ni2 on H282C- - mENaCs without MTSET is the same as in Fig.
D shows one of the possible reasons why MTSET inhibited H282C- - mENaC without effect on - - H239C mENaC.
Attachment of MTSET to sulfhydryl group of H282C would prevent Ni2 from entering the binding site to cause inhibition of the channel currents.
For 5 0109 several mutations, such as R280H, H282C, and H239C, the high affinity component of the Ni2 dose-response curve appeared to be shifted more than the low affinity component (Figs.
W278H
protein
substitution
true positive
Q61180
Three other mutations b ( W278H, Y283H, and N285H) slightly decreased Ni2 lock of the Na currents, whereas three other mutations ( Y279H, F281H, and I284H) did not significantly change Ni2 block (Fig.
R280H
protein
substitution
true positive
Q61180
In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/ H282 (referred to as His279 282) and H282/Y283H/I284H/ N285H (referred to as His282285), were also generated.
His279 282 and His282285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H.
G, proposed Ni2 coordination models for WT (left), R280H (middle), and R280H/ H239R mENaCs (right).
Y279H
protein
substitution
true positive
Q61180
In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/ H282 (referred to as His279 282) and H282/Y283H/I284H/ N285H (referred to as His282285), were also generated.
His279 282 and His282285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H.
H319R
protein
substitution
Q9WU38
true positive
Channel n Ki mM Hill coefficient R2 -H381R- - H319R- - H338R - Q220H- 6 6 8 6 6 0.58 0.73 0.82 0.97 1.03 0.09 0.15 0.12 0.22 0.10a 0.37 0.42 0.33 0.39 0.33 0.01 0.02 0.02 0.03 0.01 0.9545 0.9891 0.9846 0.9895 0.9529 0.0116 0.0022 0.0025 0.0022 0.0054 a p 0.01 from Student's t tests between WT and the mutant channel.
H239R
protein
substitution
Q9WU39
true positive
The mutation H282D or double mutations H282R/ H239R eliminated Ni2 block.
The mutations H239C, H239R, and H239D significantly attenuated Ni2 inhibition (Fig.
The double mutations ( H282R and H239R) eliminated Ni2 inhibition of Na currents (Fig.
Interestingly, double mutations ( R280H and H239R) shifted the high affinity component in the doseresponse curve to the right compared with R280H- - but to the left compared with WT mENaCs.
A, dose-response curves of Ni2 on - - H239R ( ), - - H239D (q), and - - H239C (OE) mENaCs were generated by fitting the data with the one-site equation (dashed lines) and twosite equation (solid lines) as described under "Experimental Procedures." WT doseresponse curve (E) is shown for comparison.
Numbers of clamped oocytes are 6, 5, and 7 for - - H239R, - - H239D, and - H239C, respectively.
Double mutations ( H282R/ H239R) eliminated Ni2 inhibition.
A, dose response of Ni2 on amiloridesensitive Na currents was examined in oocytes expressing H282R- - H239R mENaCs.
E, dose responses of Ni2 on WT (E), R280H- - ( ), and R280H- - H239R (,) mENaCs were examined with nine concentrations of Ni2 .
G, proposed Ni2 coordination models for WT (left), R280H (middle), and R280H/ H239R mENaCs (right).
A solvent (water) replaces the eliminated His239 in R280H- - H239R channels.
Channel n A K1 mM B K2 mM R2 -R280H- R280H- - H239R a b c 8 6 7 0.58 0.57 0.48 0.02 0.02 0.01c 0.029 0.002 0.003 0.003 0.000c 0.000c 0.22 0.16 0.33 0.01 0.01a 0.01c 6.93 0.94 8.04 1.59 0.19b 1.14 0.9968 0.9939 0.9933 0.0008 0.0019 0.0009 Values are p Values are p Values are p 0.05 from Student's t test between WT and mutant channels.
Moreover, the observed Ni2 inhibition of ENaC currents was specifically eliminated by H282D mutation and by the double ( H282R/ H239R) mutation, which strongly suggests that the whole-cell current reduction is due to Ni2 interaction with ENaC rather than with other channels.
The -mutation-induced changes in Ni2 inhibition followed the order: H239R H 239D H239C, consistent with the preferential coordination of Ni2 .
The enhanced Ni2 inhibition by R280H was attenuated by mutation of His239 ( R280H- - H239R).
Substitution of His282 or His239 with cysteine or arginine produced similar shifts in the Ni2 dose-response curves, and channels with mutations at both sites ( H282R- - H239R) were Ni2 -insenb sitive.
The elimination of Ni2 nhibition by point mutation H282D, the enhanced Ni2 inhibition by R280H, and partial reversal of the enhancement by a mutation in ENaC ( H239R) also support this view.
The observations that H282D or H282R/ H239R eliminated both high and low affinity inhibition are not consistent with the possibility.
H282W
protein
substitution
true positive
Q61180
4A, all four mutations ( H282C, H282R, H282W, and H282D) dramatically ded creased Ni2 inhibition as evidenced by the shift in the Ni2 ose-response curves of the mutant channels to the right with respect to that of WT.
The changes in Ni2 inhibition observed H282W wH h these mutant channels were H282D it 282R H282C.
A, dose-response c ( urves of external Ni2 on H282R- ), H282D- - ( ), H282C- - (OE), and H282W- - (q) mENaCs.
Numbers of clamped oocytes are 4 for H282C- a nd H282W- - , 5 for H282R- - , and 14 for H282D- - .
Y283H
protein
substitution
true positive
Q61180
In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/ H282 (referred to as His279 282) and H282/Y283H/I284H/ N285H (referred to as His282285), were also generated.
His279 282 and His282285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H.
H282R
protein
substitution
true positive
Q61180
The mutation H282D or double mutations H282R/ H239R eliminated Ni2 block.
4A, all four mutations ( H282C, H282R, H282W, and H282D) dramatically ded creased Ni2 inhibition as evidenced by the shift in the Ni2 ose-response curves of the mutant channels to the right with respect to that of WT.
The double mutations ( H282R and H239R) eliminated Ni2 inhibition of Na currents (Fig.
A, dose-response c ( urves of external Ni2 on H282R- ), H282D- - ( ), H282C- - (OE), and H282W- - (q) mENaCs.
Numbers of clamped oocytes are 4 for H282C- a nd H282W- - , 5 for H282R- - , and 14 for H282D- - .
Double mutations ( H282R/ H239R) eliminated Ni2 inhibition.
A, dose response of Ni2 on amiloridesensitive Na currents was examined in oocytes expressing H282R- - H239R mENaCs.
Moreover, the observed Ni2 inhibition of ENaC currents was specifically eliminated by H282D mutation and by the double ( H282R/ H239R) mutation, which strongly suggests that the whole-cell current reduction is due to Ni2 interaction with ENaC rather than with other channels.
Substitution of His282 or His239 with cysteine or arginine produced similar shifts in the Ni2 dose-response curves, and channels with mutations at both sites ( H282R- - H239R) were Ni2 -insenb sitive.
The observations that H282D or H282R/ H239R eliminated both high and low affinity inhibition are not consistent with the possibility.
H381R
protein
substitution
true positive
Q61180
Channel n Ki mM Hill coefficient R2 -H381R- - H319R- - H338R - Q220H- 6 6 8 6 6 0.58 0.73 0.82 0.97 1.03 0.09 0.15 0.12 0.22 0.10a 0.37 0.42 0.33 0.39 0.33 0.01 0.02 0.02 0.03 0.01 0.9545 0.9891 0.9846 0.9895 0.9529 0.0116 0.0022 0.0025 0.0022 0.0054 a p 0.01 from Student's t tests between WT and the mutant channel.
I284H
protein
substitution
true positive
Q61180
In addition, two -subunit mutants with consecutive 4-histidine tracts, Y279H/R280H/F281H/ H282 (referred to as His279 282) and H282/Y283H/I284H/ N285H (referred to as His282285), were also generated.
Three other mutations b ( W278H, Y283H, and N285H) slightly decreased Ni2 lock of the Na currents, whereas three other mutations ( Y279H, F281H, and I284H) did not significantly change Ni2 block (Fig.
His279 282 and His282285 represent two multiple mutations: Y279H/R280H/F281H and Y283H/I284H/N285H.
H239D
protein
substitution
Q9WU39
true positive
The mutations H239C, H239R, and H239D significantly attenuated Ni2 inhibition (Fig.
A, dose-response curves of Ni2 on - - H239R ( ), - - H239D (q), and - - H239C (OE) mENaCs were generated by fitting the data with the one-site equation (dashed lines) and twosite equation (solid lines) as described under "Experimental Procedures." WT doseresponse curve (E) is shown for comparison.
Numbers of clamped oocytes are 6, 5, and 7 for - - H239R, - - H239D, and - H239C, respectively.
Although we do not have direct evidence for the involvement of two His282 residues in Ni2 coordination, the loss of Ni2 block observed with H282D- - channels, compared with the modest change in Ni2 sensitivity observed with - - H239D channels, is consistent with the notion that more than one -subunit participates in the coordinated binding of Ni2 .
H239C
protein
substitution
Q9WU39
true positive
Although H282C- - channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), - - H239C channels were insensitive to MTSET.
The mutations H239C, H239R, and H239D significantly attenuated Ni2 inhibition (Fig.
MTSET at 1 mM did not alter amiloride-sensitive Na currents in oocytes expressing WT - - mENaCs (24) or - - H239C mENaCs (Fig.
A possible explanation of why MTSET failed to inhibit - - H239C mENaC is illustrated in Fig.
A, dose-response curves of Ni2 on - - H239R ( ), - - H239D (q), and - - H239C (OE) mENaCs were generated by fitting the data with the one-site equation (dashed lines) and twosite equation (solid lines) as described under "Experimental Procedures." WT doseresponse curve (E) is shown for comparison.
Numbers of clamped oocytes are 6, 5, and 7 for - - H239R, - - H239D, and - H239C, respectively.
A, effects of MTSET on H282C- - and - - H239C mENaCs.
Open, filled, and shaded bars are the relative currents (mean S.E., n 5 for H282C- - and n 13 for - - H239C) obtained before and after MTSET and after washout of MTSET, respectively.
D shows one of the possible reasons why MTSET inhibited H282C- - mENaC without effect on - - H239C mENaC.
The SH group of H239C may be too deep for interaction with MTSET.
The -mutation-induced changes in Ni2 inhibition followed the order: H239R H 239D H239C, consistent with the preferential coordination of Ni2 .
For 5 0109 several mutations, such as R280H, H282C, and H239C, the high affinity component of the Ni2 dose-response curve appeared to be shifted more than the low affinity component (Figs.
H338R
protein
substitution
Q9WU39
true positive
Channel n Ki mM Hill coefficient R2 -H381R- - H319R- - H338R - Q220H- 6 6 8 6 6 0.58 0.73 0.82 0.97 1.03 0.09 0.15 0.12 0.22 0.10a 0.37 0.42 0.33 0.39 0.33 0.01 0.02 0.02 0.03 0.01 0.9545 0.9891 0.9846 0.9895 0.9529 0.0116 0.0022 0.0025 0.0022 0.0054 a p 0.01 from Student's t tests between WT and the mutant channel.
11696608
full text
11159396
full text
E462R
protein
substitution
true positive
P15381
The reverse mutation E462R in the L-type 1C (CaV1.2) produced channels with inactivation properties comparable to 1E R378E.
Point mutations E462R in 1C and its counterpart R387E in 1E channels were herein shown to significantly influence both the kinetics and the voltage dependence of inactivation.
The cRNA concentration of the 1-subunit was generally adjusted to yield whole-cell peak currents in the 15- A range; hence RNA concentration coding for the 1E wild-type and mutant channels was established at the lowest end of this range whereas 1C wild-type and 1C E462R channels were measured after injection with the highest concentration possible.
Current traces recorded under the same conditions for the modified 1C (XhoI) channel (used to produce the 1C E462R mutant) are shown for comparison.
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
The reverse mutation E462R triggers faster inactivation kinetics in 1C (CaV1.2) The alignment shown in Fig.
To compare the inactivation properties of 1E R378E and 1C E462R under our experimental conditions, we performed the 1C E462R mutation and expressed it in Xenopus oocytes.
7 A shows whole-cell current traces for 1E, 1C E462R, 1E R378E, and 1C that were scaled and superimposed at membrane potentials between 10 and 10 mV.
7 B, 1C E462R and 1E R378E inactivated with a similar time course that turned out to be intermediary between the fast 1E and the slow 1C channel at all voltages with r300 ratios of 0.3 in both cases.
As seen, the inactivation kinetics for the modified 1C (XhoI) channel used for making 1C E462R were compiled and were found to be similar to the wild-type 1C channel.
As inactivation kinetics of 1C are exquisitely sensitive upon the current density, it should be pointed out that the current density for 1C E462R was in average smaller than for 1C (XhoI), ruling out current density as a critical factor for the faster inactivation kinetics (Table 1).
The voltage dependence of inactivation was next studied for mutant 1C E462R (5-s prepulses) and compared with 1E, 1E R378E, and 1C (XhoI) (Fig.
As compared with the 1C (XhoI) channel, the inactivation data points for 1C E462R were shifted to the left by 10 mV, but they remained significantly more positive than the inactivation curve for 1E R378E.
Furthermore, the slope of the fit was much steeper for 1C E462R than for any other mutant tested in this study.
Given the fact that the I-II linker could also be involved in calcium-dependent inactivation (Adams and Tanabe, 1997), the inactivation kinetics of 1C E462R were also measured in the presence of 10 mM Ca2 .
Like the wildtype 1C, 1C E462R was found to inactivate significantly faster in the presence of Ca2 ions.
taken to examine the possibility that the altered inactivation kinetics of 1E R378E and 1C E462R were secondary to a modification in the coupling between 3 and 1-subunits as all previous experiments were performed in the presence of a full complement of auxiliary 2b - and 3-subunits.
Mutant 1E R378E and 1C E462R were expressed in Xenopus oocytes in the presence ( 2b / 3) and in the absence of 3 with only 2b as ancillary subunit (Fig.
The presence of 3 induced a leftward shift of the peak voltage by 10 mV for both 1C E462R and 1E R378E (results not shown), which is similar to what has been reported before for the wild-type channels (Parent et al., 1997).
The voltage dependence of inactivation estimated from isochronal measurements at 5 s was shifted to the left in the presence of 3 with E0.5 11 2 mV (n 4) ( 3) and E0.5 26 2 mV (n 6) ( 3) for 1C E462R and E0.5 26 1 mV (n 3) ( 3) and E0.5 44 1 mV (n 3) ( 3) for 1E R378E (results not shown).
The time courses of inactivation for 1C E462R and 1E R378E remained comparable to each other whether it was measured in the absence (left panel) or in the presence of 3 (right panel) under all conditions except at 10 mV in the absence of 3 as it can be inferred from the r300 ratio analysis (lower left panel).
Hence, the inactivation kinetics remained similar for 1E R378E and 1C E462R in the absence of 3.
The AID motif is composed of a stretch of 18 amino acids located about at the 5 end of the I-II linker that reads QQXEXXLXGYX- -Subunit regulation is preserved in 1E R378E and 1C E462R channel mutants As -subunits are known to modulate the inactivation kinetics of the 1-subunit and to bind to the AID site (Pragnell et al., 1994), the next series of experiments was underBiophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation 223 FIGURE 6 (A) The voltage dependence of inactivation was estimated from the relative tail currents recorded after a 5-s pulse applied between 100 and 50 mV (10-mV steps).
The rates of 1C E462R and 1E R378E inactivation were both significantly altered and were almost indistinguishable at 20 mV.
FIGURE 7 (A) Whole-cell currents obtained in the presence of 10 mM Ba2 for 1E wt, 1C E462R, 1E R378E, and 1C wt were scaled and superimposed at the voltages of 10 (left), 0 (middle), and 10 mV (right).
Mutant 1C E462R and 1E R378E inactivated following a time course intermediary between 1E and 1C at all membrane potentials.
(B) The mean r300 ratios calculated for the same channels are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E R378E (dark gray), 1E E462R 1 (white), 1C (XhoI) (gray), and 1C wt (hatched) from left to right.
The r300 ratios went from 0.05 0.02 at 10 mV and 0.02 0 at 20 mV (n 7) for 1E, from 0.33 0.04 at 10 mV to 0.27 0.03 at 20 mV (n 14) for 1E R378E, from 0.39 0.04 at 10 mV to 0.26 0.02 at 20 mV (n 6) for 1C E462R, from 0.68 0.03 at 10 mV to 0.69 0.04 at 20 mV (n 6) for 1C (XhoI), and from 0.69 0.01 at 10 mV to 0.71 0 .02 at 20 mV (n 9) for 1C wt.
The estimated mid-potential of inactivation (E0.5) for 1C E462R ( ) was more negative than for the 1C (XhoI) construct (F) but more positive than for 1E R378E () and 1E wt (f).
Glycine residues are known AID Mutations in Voltage-Dependent Inactivation 225 FIGURE 8 3-Subunit modulation of mutants 1E R378E and 1C E462R.
(A) The 1E R378E and 1C E462R mutants were expressed in Xenopus oocytes in the presence of 2b (left) and 2b / 3 subunits (right).
Whole-cell currents peaked at 10 1 mV (n 4) for 1C E462R/ 2b and 1 1 mV (n 7) for 1C E462R/ 2b / 3, at 10 1 mV (n 3) for 1E R378E/ 2b , and at 0 0.2 mV (n 3) for 1E R378E/ 2b / 3.
(B) At 10 mV, r300 ( ent from 0.21 0.02 for 1E/ 2b w n 3) to 0.01 0.005 for 1E/ 2b / 3 (n 6), from 0.54 0.08 for 1E R378E/ 2b (n 3) to 0.27 0.03 for 1E R378E/ 2b / 3 (n 9), and from 0.65 0.05 for 0 1C E462R/ 2b (n 4) to 0.27 6.02 for 1C E462R/ 2b / 3 (n ).
By investigating the inactivation properties of 1E R378E and 1C E462R in the presence and in the absence of 3, we showed that the R-to-E mutation at the nonconserved position 5 of the AID motif failed to prevent -subunit modulation.
These data hence confirmed that the changes in the inactivation kinetics observed in 1E R378E and 1C E462R were intrinsically determined by changes in the 1-subunit.
R378Q
protein
substitution
true positive
Q15878
R378K behaved like the wild-type 1E whereas R378Q displayed intermediate inactivation kinetics.
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
4 shows the family of wholecell Ba2 current traces of mutants R378K, R378Q, R378A, R378G, R378D, and R378E after expression in Xenopus oocytes.
Mutant 1E R378K displayed the fastest inactivation kinetics closely followed by R378A and R378Q.
kinetics tended to get faster with membrane depolarization especially for mutants R378Q, R378G, R378D, and R378E.
For R378Q, the r300 ratio was significantly (p 0.05) different from R378K at 10 mV, but these differences were attenuated at 20 mV.
Only R378Q appeared to activate at membrane potentials slightly more positive than the wild-type 1E channel.
Mutations at position 378 affect voltagedependent inactivation of the human 1E (CaV2.3) The next series of experiments was undertaken to evaluate whether the charge of the side-chain at position 378 played AID Mutations in Voltage-Dependent Inactivation 221 FIGURE 4 Mutants 1E R378K, 1E R378Q, and 1E R378A (A) and 1E R378G, 1E R378D, and 1E R378E (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Typical current traces are shown for the wild-type 1E and mutants R378K, R378Q, and R378E with an example of the voltage protocol used.
In contrast, inactivation data points are shifted to the right for mutants 1E R378A, R378Q, R378G, R378D, and R378E and lay halfway between 1C and 1E.
The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (black), 1E R378K (light gray), 1E R378A (dark gray), 1E R378Q (white), 1E R378G (gray), 1E R378D (hatched), and 1E R378E (cross-hatched) from left to right.
At 10 mV, the r300 ratios for 1E R378K and 1E R378A were not significantly 0ifferent from 1E wt whereas those for 1E R378Q were different at p d .05 and those for 1E R378G, 1E R378D, and 1E R378E were significantly different at p 0.002.
The r300 ratios went from 0.04 0.01 0t 10 mV and 0.05 0.02 at 20 mV (n 7) for R378K, from 0.05 a .01 at 10 mV to 0.06 0.02 at 20 mV (n 4) for R378A, from 0.13 0.03 at 10 mV to 0.08 0.02 at 20 mV (n 7) for R378Q, from 0.27 0.03 at 10 mV and 0.21 0.01 at 20 mV (n 7) for R378G, from 0.25 0.03 at 10 mV to 0.19 0.02 at 20 mV (n 14) for R378D, and from 0.28 0.03 at 10 mV to 0.23 0.03 at 20 mV (n 14) for R378E as compared with 0.05 0.02 at 10 mV and 0.02 0 .01 at 20 mV (n 17) for 1E.
The activation potentials were comparable for all mutants although they increased slightly from 1E R378A 1E R378G 1E wt 1E R378K 1E R378D 1E R378E 1E R378Q.
Typical current traces for 1E wt, 1E R378K, 1E R378Q, and 1E R378E are shown from left to right.
The estimated mid-potentials of inactivation (E0.5) were comparable for 1E wt (f) and for 1E R378K ( ) whereas the mid-potentials of inactivation were shifted to the right for 1E R378A (*); 1E R378G (,), 1E R378Q (OE); 1E R378D ( ), and 1E R378E ().
A series of mutations at the R378 position indicated that the net charge carried by the sidechain could play a role in the inactivation kinetics, although the net charge carried by the residue could not explain by itself the effects of R378A, R378G, and R378Q (see below).
Experiments were undertaken to investigate the role of electrostatic interaction in the inactivation kinetics of 1E with additional mutants R378K (positive), R378A (nonpolar and neutral), R378G (polar but Biophysical Journal 80(1) 215228 neutral), R378Q (polar but neutral), and R378D (negative).
However, such an interpretation falls short of explaining the behavior of neutral mutants R378A, R378G, and R378Q.
R378Q displayed intermediary inactivation kinetics at 10 mV but tended to inactivate like R378K at 20 mV.
Hence, there was no simple correlation between the inactivation kinetics of R378 mutants (R378E, R378D, R378K, R378A, R378Q, and R378G) and any single physicochemical property (charge, polarity, hydropathicity, and hydrophilicity).
The intermediary behavior of R378Q could be partly explained by the relative polarity of its side-chain as compared with R378A.
N381K
protein
substitution
true positive
Q15878
The quintuple mutant 1E N381K R384L A385D D388T K389Q (NRADK-KLDTQ) inactivated like the wild-type 1E.
Furthermore, a quintuple mutant made in the same region, 1E N381K K R384L A385D D388T 389Q, failed to affect either kinetics or voltage dependence of inactivation.
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
A385D
protein
substitution
true positive
Q15878
The quintuple mutant 1E N381K R384L A385D D388T K389Q (NRADK-KLDTQ) inactivated like the wild-type 1E.
Furthermore, a quintuple mutant made in the same region, 1E N381K K R384L A385D D388T 389Q, failed to affect either kinetics or voltage dependence of inactivation.
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
K389Q
protein
substitution
true positive
Q15878
The quintuple mutant 1E N381K R384L A385D D388T K389Q (NRADK-KLDTQ) inactivated like the wild-type 1E.
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
It should be noted that the milder K389Q mutation achieved within the quadruple and the quintuple mutants produced no significant effect on inactivation (Fig.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
R378E
protein
substitution
true positive
Q15878
When co-injected with 3, R378E inactivated with inact 538 54 ms (n 14) as compared with 74 ms (n 21) for 1E (p 0.001) with a mid-potential of inactivation E0.5 44 2 mV (n 10) for R378E as compared with E0.5 64 3 mV (n 9) for 1E.
The reverse mutation E462R in the L-type 1C (CaV1.2) produced channels with inactivation properties comparable to 1E R378E.
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
In contrast, a single point mutation at position R378 (position 5 in AID) produced whole-cell currents with significantly slower inactivation kinetics with only 72 4% (n 13) of the R378E currents being inactivated under the same conditions.
Whole-cell current density was lower for the fast inactivating mutant 1E NRA-KLD whereas the largest currents were generally recorded for 1E R378E.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Inactivation kinetics were slower in mutants K389E and R378E.
Whole-cell currents peaked at 4 2 mV (n 7) for 1E; 3 3 mV (n 3) for NRA-KLD; 5 3 mV (n 4) for NRAK-KLDQ; 3 3 mV (n 3) for NRADK-KLDTQ; 3 2 mV (n 9) for 1E R378E, and 4 2 mV (n 9) for 1C (XhoI).
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
In contrast, the r300 ratios were significantly higher for 1E R378E (p 0.002) and 1E K389E (p 0.05) as compared with 1E.
Whereas inactivation kinetics were significantly slower for 1E K389E and R378E, both mutants were found to activate in a range of potentials not significantly different than 1E wt (Table 1).
3 C shows a family of isochronal inactivation data for the wild-type 1E channel, NRA-KLD, NRAK-KLDQ (results not shown), NRADK-KLDTQ, K389E, R378E, and 1C wt.
Only mutant R378E experienced a significant shift in its voltage dependence of inactivation toward more positive potentials as compared with the wild-type 1E with a E0.5 44 mV.
This change in the voltage dependence of inactivation of 1E R378E occurred without any significant shift in the voltage dependence of activation (Table 1).
4 shows the family of wholecell Ba2 current traces of mutants R378K, R378Q, R378A, R378G, R378D, and R378E after expression in Xenopus oocytes.
At 10 mV, the rate of inactivation ranked as follows (from R e fastest to the slowest) 1E wt th R378K R378A 378Q R378G R378D R378E as seen by the r300 analysis shown in Fig.
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
The r300 ratios were also significantly different between 1E wt and 1E R378E (p 0.002) with values from 0.33 0.04 at 10 mV to 0.27 0.03 (n 14) at 20 mV for R378E.
The activation potentials were comparable for 1E wt, NRA-KLD, NRAK-KLDQ, NRADK-KLDTQ (results not shown), 1E K389E, and 1E R378E.
In contrast, the mid-potential of inactivation for R378E was 44 s mV (n 10).
kinetics tended to get faster with membrane depolarization especially for mutants R378Q, R378G, R378D, and R378E.
R378A, a small and neutral residue, behaved like R378K, but R378G, which is also a small and neutral residue, behaved like negatively charged mutants R378D and R378E.
Furthermore, the activation potentials E0.5 for mutants R378E, R378D, R378G, R378A, and R378K were comparable to the E0.5 for the wild-type 1E channel.
Mutations at position 378 affect voltagedependent inactivation of the human 1E (CaV2.3) The next series of experiments was undertaken to evaluate whether the charge of the side-chain at position 378 played AID Mutations in Voltage-Dependent Inactivation 221 FIGURE 4 Mutants 1E R378K, 1E R378Q, and 1E R378A (A) and 1E R378G, 1E R378D, and 1E R378E (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Inactivation kinetics appeared slower in mutants R378G, R378D, and R378E.
Typical current traces are shown for the wild-type 1E and mutants R378K, R378Q, and R378E with an example of the voltage protocol used.
In contrast, inactivation data points are shifted to the right for mutants 1E R378A, R378Q, R378G, R378D, and R378E and lay halfway between 1C and 1E.
Mid-potentials of inactivation ranged from E0.5 52 mV for R378G to E0.5 44 mV for R378E.
To compare the inactivation properties of 1E R378E and 1C E462R under our experimental conditions, we performed the 1C E462R mutation and expressed it in Xenopus oocytes.
7 A shows whole-cell current traces for 1E, 1C E462R, 1E R378E, and 1C that were scaled and superimposed at membrane potentials between 10 and 10 mV.
7 B, 1C E462R and 1E R378E inactivated with a similar time course that turned out to be intermediary between the fast 1E and the slow 1C channel at all voltages with r300 ratios of 0.3 in both cases.
The voltage dependence of inactivation was next studied for mutant 1C E462R (5-s prepulses) and compared with 1E, 1E R378E, and 1C (XhoI) (Fig.
As compared with the 1C (XhoI) channel, the inactivation data points for 1C E462R were shifted to the left by 10 mV, but they remained significantly more positive than the inactivation curve for 1E R378E.
The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (black), 1E R378K (light gray), 1E R378A (dark gray), 1E R378Q (white), 1E R378G (gray), 1E R378D (hatched), and 1E R378E (cross-hatched) from left to right.
At 10 mV, the r300 ratios for 1E R378K and 1E R378A were not significantly 0ifferent from 1E wt whereas those for 1E R378Q were different at p d .05 and those for 1E R378G, 1E R378D, and 1E R378E were significantly different at p 0.002.
The r300 ratios went from 0.04 0.01 0t 10 mV and 0.05 0.02 at 20 mV (n 7) for R378K, from 0.05 a .01 at 10 mV to 0.06 0.02 at 20 mV (n 4) for R378A, from 0.13 0.03 at 10 mV to 0.08 0.02 at 20 mV (n 7) for R378Q, from 0.27 0.03 at 10 mV and 0.21 0.01 at 20 mV (n 7) for R378G, from 0.25 0.03 at 10 mV to 0.19 0.02 at 20 mV (n 14) for R378D, and from 0.28 0.03 at 10 mV to 0.23 0.03 at 20 mV (n 14) for R378E as compared with 0.05 0.02 at 10 mV and 0.02 0 .01 at 20 mV (n 17) for 1E.
The activation potentials were comparable for all mutants although they increased slightly from 1E R378A 1E R378G 1E wt 1E R378K 1E R378D 1E R378E 1E R378Q.
taken to examine the possibility that the altered inactivation kinetics of 1E R378E and 1C E462R were secondary to a modification in the coupling between 3 and 1-subunits as all previous experiments were performed in the presence of a full complement of auxiliary 2b - and 3-subunits.
Mutant 1E R378E and 1C E462R were expressed in Xenopus oocytes in the presence ( 2b / 3) and in the absence of 3 with only 2b as ancillary subunit (Fig.
The presence of 3 induced a leftward shift of the peak voltage by 10 mV for both 1C E462R and 1E R378E (results not shown), which is similar to what has been reported before for the wild-type channels (Parent et al., 1997).
The voltage dependence of inactivation estimated from isochronal measurements at 5 s was shifted to the left in the presence of 3 with E0.5 11 2 mV (n 4) ( 3) and E0.5 26 2 mV (n 6) ( 3) for 1C E462R and E0.5 26 1 mV (n 3) ( 3) and E0.5 44 1 mV (n 3) ( 3) for 1E R378E (results not shown).
The time courses of inactivation for 1C E462R and 1E R378E remained comparable to each other whether it was measured in the absence (left panel) or in the presence of 3 (right panel) under all conditions except at 10 mV in the absence of 3 as it can be inferred from the r300 ratio analysis (lower left panel).
Hence, the inactivation kinetics remained similar for 1E R378E and 1C E462R in the absence of 3.
The AID motif is composed of a stretch of 18 amino acids located about at the 5 end of the I-II linker that reads QQXEXXLXGYX- -Subunit regulation is preserved in 1E R378E and 1C E462R channel mutants As -subunits are known to modulate the inactivation kinetics of the 1-subunit and to bind to the AID site (Pragnell et al., 1994), the next series of experiments was underBiophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation 223 FIGURE 6 (A) The voltage dependence of inactivation was estimated from the relative tail currents recorded after a 5-s pulse applied between 100 and 50 mV (10-mV steps).
Typical current traces for 1E wt, 1E R378K, 1E R378Q, and 1E R378E are shown from left to right.
The estimated mid-potentials of inactivation (E0.5) were comparable for 1E wt (f) and for 1E R378K ( ) whereas the mid-potentials of inactivation were shifted to the right for 1E R378A (*); 1E R378G (,), 1E R378Q (OE); 1E R378D ( ), and 1E R378E ().
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
Of the two mutants, only the voltage dependence of inactivation of 1E R378E was affected with 20-mV shift in its mid-potential of inactivation as compared with 1E wt.
Alterations in the inactivation properties occurred without any significant difference in the activation properties, hence suggesting that the inactivated state was intrinsically mod- ified by the R378E mutation.
The rates of 1C E462R and 1E R378E inactivation were both significantly altered and were almost indistinguishable at 20 mV.
FIGURE 7 (A) Whole-cell currents obtained in the presence of 10 mM Ba2 for 1E wt, 1C E462R, 1E R378E, and 1C wt were scaled and superimposed at the voltages of 10 (left), 0 (middle), and 10 mV (right).
Mutant 1C E462R and 1E R378E inactivated following a time course intermediary between 1E and 1C at all membrane potentials.
(B) The mean r300 ratios calculated for the same channels are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E R378E (dark gray), 1E E462R 1 (white), 1C (XhoI) (gray), and 1C wt (hatched) from left to right.
The r300 ratios went from 0.05 0.02 at 10 mV and 0.02 0 at 20 mV (n 7) for 1E, from 0.33 0.04 at 10 mV to 0.27 0.03 at 20 mV (n 14) for 1E R378E, from 0.39 0.04 at 10 mV to 0.26 0.02 at 20 mV (n 6) for 1C E462R, from 0.68 0.03 at 10 mV to 0.69 0.04 at 20 mV (n 6) for 1C (XhoI), and from 0.69 0.01 at 10 mV to 0.71 0 .02 at 20 mV (n 9) for 1C wt.
The estimated mid-potential of inactivation (E0.5) for 1C E462R ( ) was more negative than for the 1C (XhoI) construct (F) but more positive than for 1E R378E () and 1E wt (f).
In contrast, negatively charged mutants R378D and R378E showed the slowest inactivation properties.
R378G produced, on the other hand, slow inactivation kinetics comparable to R378D and R378E at all membrane potentials.
Hence, there was no simple correlation between the inactivation kinetics of R378 mutants (R378E, R378D, R378K, R378A, R378Q, and R378G) and any single physicochemical property (charge, polarity, hydropathicity, and hydrophilicity).
Glycine residues are known AID Mutations in Voltage-Dependent Inactivation 225 FIGURE 8 3-Subunit modulation of mutants 1E R378E and 1C E462R.
(A) The 1E R378E and 1C E462R mutants were expressed in Xenopus oocytes in the presence of 2b (left) and 2b / 3 subunits (right).
Whole-cell currents peaked at 10 1 mV (n 4) for 1C E462R/ 2b and 1 1 mV (n 7) for 1C E462R/ 2b / 3, at 10 1 mV (n 3) for 1E R378E/ 2b , and at 0 0.2 mV (n 3) for 1E R378E/ 2b / 3.
(B) At 10 mV, r300 ( ent from 0.21 0.02 for 1E/ 2b w n 3) to 0.01 0.005 for 1E/ 2b / 3 (n 6), from 0.54 0.08 for 1E R378E/ 2b (n 3) to 0.27 0.03 for 1E R378E/ 2b / 3 (n 9), and from 0.65 0.05 for 0 1C E462R/ 2b (n 4) to 0.27 6.02 for 1C E462R/ 2b / 3 (n ).
By investigating the inactivation properties of 1E R378E and 1C E462R in the presence and in the absence of 3, we showed that the R-to-E mutation at the nonconserved position 5 of the AID motif failed to prevent -subunit modulation.
These data hence confirmed that the changes in the inactivation kinetics observed in 1E R378E and 1C E462R were intrinsically determined by changes in the 1-subunit.
R378D
protein
substitution
true positive
Q15878
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
4 shows the family of wholecell Ba2 current traces of mutants R378K, R378Q, R378A, R378G, R378D, and R378E after expression in Xenopus oocytes.
At 10 mV, the rate of inactivation ranked as follows (from R e fastest to the slowest) 1E wt th R378K R378A 378Q R378G R378D R378E as seen by the r300 analysis shown in Fig.
kinetics tended to get faster with membrane depolarization especially for mutants R378Q, R378G, R378D, and R378E.
R378A, a small and neutral residue, behaved like R378K, but R378G, which is also a small and neutral residue, behaved like negatively charged mutants R378D and R378E.
Furthermore, the activation potentials E0.5 for mutants R378E, R378D, R378G, R378A, and R378K were comparable to the E0.5 for the wild-type 1E channel.
Mutations at position 378 affect voltagedependent inactivation of the human 1E (CaV2.3) The next series of experiments was undertaken to evaluate whether the charge of the side-chain at position 378 played AID Mutations in Voltage-Dependent Inactivation 221 FIGURE 4 Mutants 1E R378K, 1E R378Q, and 1E R378A (A) and 1E R378G, 1E R378D, and 1E R378E (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Inactivation kinetics appeared slower in mutants R378G, R378D, and R378E.
In contrast, inactivation data points are shifted to the right for mutants 1E R378A, R378Q, R378G, R378D, and R378E and lay halfway between 1C and 1E.
The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (black), 1E R378K (light gray), 1E R378A (dark gray), 1E R378Q (white), 1E R378G (gray), 1E R378D (hatched), and 1E R378E (cross-hatched) from left to right.
At 10 mV, the r300 ratios for 1E R378K and 1E R378A were not significantly 0ifferent from 1E wt whereas those for 1E R378Q were different at p d .05 and those for 1E R378G, 1E R378D, and 1E R378E were significantly different at p 0.002.
The r300 ratios went from 0.04 0.01 0t 10 mV and 0.05 0.02 at 20 mV (n 7) for R378K, from 0.05 a .01 at 10 mV to 0.06 0.02 at 20 mV (n 4) for R378A, from 0.13 0.03 at 10 mV to 0.08 0.02 at 20 mV (n 7) for R378Q, from 0.27 0.03 at 10 mV and 0.21 0.01 at 20 mV (n 7) for R378G, from 0.25 0.03 at 10 mV to 0.19 0.02 at 20 mV (n 14) for R378D, and from 0.28 0.03 at 10 mV to 0.23 0.03 at 20 mV (n 14) for R378E as compared with 0.05 0.02 at 10 mV and 0.02 0 .01 at 20 mV (n 17) for 1E.
The activation potentials were comparable for all mutants although they increased slightly from 1E R378A 1E R378G 1E wt 1E R378K 1E R378D 1E R378E 1E R378Q.
The estimated mid-potentials of inactivation (E0.5) were comparable for 1E wt (f) and for 1E R378K ( ) whereas the mid-potentials of inactivation were shifted to the right for 1E R378A (*); 1E R378G (,), 1E R378Q (OE); 1E R378D ( ), and 1E R378E ().
Experiments were undertaken to investigate the role of electrostatic interaction in the inactivation kinetics of 1E with additional mutants R378K (positive), R378A (nonpolar and neutral), R378G (polar but Biophysical Journal 80(1) 215228 neutral), R378Q (polar but neutral), and R378D (negative).
In contrast, negatively charged mutants R378D and R378E showed the slowest inactivation properties.
R378G produced, on the other hand, slow inactivation kinetics comparable to R378D and R378E at all membrane potentials.
Hence, there was no simple correlation between the inactivation kinetics of R378 mutants (R378E, R378D, R378K, R378A, R378Q, and R378G) and any single physicochemical property (charge, polarity, hydropathicity, and hydrophilicity).
R378G
protein
substitution
true positive
Q15878
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
4 shows the family of wholecell Ba2 current traces of mutants R378K, R378Q, R378A, R378G, R378D, and R378E after expression in Xenopus oocytes.
At 10 mV, the rate of inactivation ranked as follows (from R e fastest to the slowest) 1E wt th R378K R378A 378Q R378G R378D R378E as seen by the r300 analysis shown in Fig.
kinetics tended to get faster with membrane depolarization especially for mutants R378Q, R378G, R378D, and R378E.
R378A, a small and neutral residue, behaved like R378K, but R378G, which is also a small and neutral residue, behaved like negatively charged mutants R378D and R378E.
Furthermore, the activation potentials E0.5 for mutants R378E, R378D, R378G, R378A, and R378K were comparable to the E0.5 for the wild-type 1E channel.
Mutations at position 378 affect voltagedependent inactivation of the human 1E (CaV2.3) The next series of experiments was undertaken to evaluate whether the charge of the side-chain at position 378 played AID Mutations in Voltage-Dependent Inactivation 221 FIGURE 4 Mutants 1E R378K, 1E R378Q, and 1E R378A (A) and 1E R378G, 1E R378D, and 1E R378E (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Inactivation kinetics appeared slower in mutants R378G, R378D, and R378E.
In contrast, inactivation data points are shifted to the right for mutants 1E R378A, R378Q, R378G, R378D, and R378E and lay halfway between 1C and 1E.
Mid-potentials of inactivation ranged from E0.5 52 mV for R378G to E0.5 44 mV for R378E.
The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (black), 1E R378K (light gray), 1E R378A (dark gray), 1E R378Q (white), 1E R378G (gray), 1E R378D (hatched), and 1E R378E (cross-hatched) from left to right.
At 10 mV, the r300 ratios for 1E R378K and 1E R378A were not significantly 0ifferent from 1E wt whereas those for 1E R378Q were different at p d .05 and those for 1E R378G, 1E R378D, and 1E R378E were significantly different at p 0.002.
The r300 ratios went from 0.04 0.01 0t 10 mV and 0.05 0.02 at 20 mV (n 7) for R378K, from 0.05 a .01 at 10 mV to 0.06 0.02 at 20 mV (n 4) for R378A, from 0.13 0.03 at 10 mV to 0.08 0.02 at 20 mV (n 7) for R378Q, from 0.27 0.03 at 10 mV and 0.21 0.01 at 20 mV (n 7) for R378G, from 0.25 0.03 at 10 mV to 0.19 0.02 at 20 mV (n 14) for R378D, and from 0.28 0.03 at 10 mV to 0.23 0.03 at 20 mV (n 14) for R378E as compared with 0.05 0.02 at 10 mV and 0.02 0 .01 at 20 mV (n 17) for 1E.
The activation potentials were comparable for all mutants although they increased slightly from 1E R378A 1E R378G 1E wt 1E R378K 1E R378D 1E R378E 1E R378Q.
The estimated mid-potentials of inactivation (E0.5) were comparable for 1E wt (f) and for 1E R378K ( ) whereas the mid-potentials of inactivation were shifted to the right for 1E R378A (*); 1E R378G (,), 1E R378Q (OE); 1E R378D ( ), and 1E R378E ().
A series of mutations at the R378 position indicated that the net charge carried by the sidechain could play a role in the inactivation kinetics, although the net charge carried by the residue could not explain by itself the effects of R378A, R378G, and R378Q (see below).
Experiments were undertaken to investigate the role of electrostatic interaction in the inactivation kinetics of 1E with additional mutants R378K (positive), R378A (nonpolar and neutral), R378G (polar but Biophysical Journal 80(1) 215228 neutral), R378Q (polar but neutral), and R378D (negative).
However, such an interpretation falls short of explaining the behavior of neutral mutants R378A, R378G, and R378Q.
R378G produced, on the other hand, slow inactivation kinetics comparable to R378D and R378E at all membrane potentials.
Hence, there was no simple correlation between the inactivation kinetics of R378 mutants (R378E, R378D, R378K, R378A, R378Q, and R378G) and any single physicochemical property (charge, polarity, hydropathicity, and hydrophilicity).
However, R378G, which also bears a neutral but slightly polar residue at the same position, diverged from that prediction.
R378A
protein
substitution
true positive
Q15878
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
4 shows the family of wholecell Ba2 current traces of mutants R378K, R378Q, R378A, R378G, R378D, and R378E after expression in Xenopus oocytes.
Mutant 1E R378K displayed the fastest inactivation kinetics closely followed by R378A and R378Q.
At 10 mV, the rate of inactivation ranked as follows (from R e fastest to the slowest) 1E wt th R378K R378A 378Q R378G R378D R378E as seen by the r300 analysis shown in Fig.
The r300 ratios for the neutral residue R378A were noticeably similar to R378K and to the wild-type 1E channel at all membrane potentials.
R378A, a small and neutral residue, behaved like R378K, but R378G, which is also a small and neutral residue, behaved like negatively charged mutants R378D and R378E.
Furthermore, the activation potentials E0.5 for mutants R378E, R378D, R378G, R378A, and R378K were comparable to the E0.5 for the wild-type 1E channel.
Mutations at position 378 affect voltagedependent inactivation of the human 1E (CaV2.3) The next series of experiments was undertaken to evaluate whether the charge of the side-chain at position 378 played AID Mutations in Voltage-Dependent Inactivation 221 FIGURE 4 Mutants 1E R378K, 1E R378Q, and 1E R378A (A) and 1E R378G, 1E R378D, and 1E R378E (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
In contrast, inactivation data points are shifted to the right for mutants 1E R378A, R378Q, R378G, R378D, and R378E and lay halfway between 1C and 1E.
The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (black), 1E R378K (light gray), 1E R378A (dark gray), 1E R378Q (white), 1E R378G (gray), 1E R378D (hatched), and 1E R378E (cross-hatched) from left to right.
At 10 mV, the r300 ratios for 1E R378K and 1E R378A were not significantly 0ifferent from 1E wt whereas those for 1E R378Q were different at p d .05 and those for 1E R378G, 1E R378D, and 1E R378E were significantly different at p 0.002.
The r300 ratios went from 0.04 0.01 0t 10 mV and 0.05 0.02 at 20 mV (n 7) for R378K, from 0.05 a .01 at 10 mV to 0.06 0.02 at 20 mV (n 4) for R378A, from 0.13 0.03 at 10 mV to 0.08 0.02 at 20 mV (n 7) for R378Q, from 0.27 0.03 at 10 mV and 0.21 0.01 at 20 mV (n 7) for R378G, from 0.25 0.03 at 10 mV to 0.19 0.02 at 20 mV (n 14) for R378D, and from 0.28 0.03 at 10 mV to 0.23 0.03 at 20 mV (n 14) for R378E as compared with 0.05 0.02 at 10 mV and 0.02 0 .01 at 20 mV (n 17) for 1E.
The activation potentials were comparable for all mutants although they increased slightly from 1E R378A 1E R378G 1E wt 1E R378K 1E R378D 1E R378E 1E R378Q.
The estimated mid-potentials of inactivation (E0.5) were comparable for 1E wt (f) and for 1E R378K ( ) whereas the mid-potentials of inactivation were shifted to the right for 1E R378A (*); 1E R378G (,), 1E R378Q (OE); 1E R378D ( ), and 1E R378E ().
A series of mutations at the R378 position indicated that the net charge carried by the sidechain could play a role in the inactivation kinetics, although the net charge carried by the residue could not explain by itself the effects of R378A, R378G, and R378Q (see below).
Experiments were undertaken to investigate the role of electrostatic interaction in the inactivation kinetics of 1E with additional mutants R378K (positive), R378A (nonpolar and neutral), R378G (polar but Biophysical Journal 80(1) 215228 neutral), R378Q (polar but neutral), and R378D (negative).
However, such an interpretation falls short of explaining the behavior of neutral mutants R378A, R378G, and R378Q.
Mutant R378A inactivated like R378K between 10 mV and 20 mV.
Hence, there was no simple correlation between the inactivation kinetics of R378 mutants (R378E, R378D, R378K, R378A, R378Q, and R378G) and any single physicochemical property (charge, polarity, hydropathicity, and hydrophilicity).
The intermediary behavior of R378Q could be partly explained by the relative polarity of its side-chain as compared with R378A.
R387E
protein
substitution
true positive
Q15878
Point mutations E462R in 1C and its counterpart R387E in 1E channels were herein shown to significantly influence both the kinetics and the voltage dependence of inactivation.
K389E
protein
substitution
true positive
Q15878
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Inactivation kinetics were slower in mutants K389E and R378E.
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
In contrast, the r300 ratios were significantly higher for 1E R378E (p 0.002) and 1E K389E (p 0.05) as compared with 1E.
Whereas inactivation kinetics were significantly slower for 1E K389E and R378E, both mutants were found to activate in a range of potentials not significantly different than 1E wt (Table 1).
3 C shows a family of isochronal inactivation data for the wild-type 1E channel, NRA-KLD, NRAK-KLDQ (results not shown), NRADK-KLDTQ, K389E, R378E, and 1C wt.
The voltage dependence of the isochronal inactivation for the first four mutant channels were comparable, with E0.5 varying from 60 mV (NRADK and K389E) to 68 mV (NRA).
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
In contrast, r300 ratios were significantly different between 1E wt and 1E K389E (p 0.01) varying from 0.16 0.02 at 10 mV to 0.10 0.01 (n 15) at 20 mV for 1E K389E.
The activation potentials were comparable for 1E wt, NRA-KLD, NRAK-KLDQ, NRADK-KLDTQ (results not shown), 1E K389E, and 1E R378E.
The voltage dependence of inactivation was not significantly different for 1E wt, NRA-KLD, NRAK-KLDQ (results not 2hown), NRADK-KLDTQ, and 1E K389E with E0.5 varying from 68 to 60 mV.
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
The mutation of a neighboring positive residue in K389E slowed the inactivation kinetics but failed to significantly influence the voltage dependence of inactivation.
D388T
protein
substitution
true positive
Q15878
The quintuple mutant 1E N381K R384L A385D D388T K389Q (NRADK-KLDTQ) inactivated like the wild-type 1E.
Furthermore, a quintuple mutant made in the same region, 1E N381K K R384L A385D D388T 389Q, failed to affect either kinetics or voltage dependence of inactivation.
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
R384L
protein
substitution
true positive
Q15878
The quintuple mutant 1E N381K R384L A385D D388T K389Q (NRADK-KLDTQ) inactivated like the wild-type 1E.
Furthermore, a quintuple mutant made in the same region, 1E N381K K R384L A385D D388T 389Q, failed to affect either kinetics or voltage dependence of inactivation.
2 shows a family of whole-cell current recordings obtained in the presence of 10 mM Ba2 for the wild-type 1E, triple-mutant N381K R384L A385D, quintuplemutant N381K R384L A385D D388T K389Q, and point mutations K389E and R378E expressed in Xenopus oocytes on the 2b and 3 auxiliary subunit background.
As seen, the rate of inactivation increased slightly with depo- FIGURE 2 The 1E wt, 1E N381K R384L A385D (NRA), and 1E N381K R384L A385D D388T K389Q (NRADK) (A) and 1E K389E, 1E R378E, and 1C (XhoI) (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
FIGURE 3 (A) The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (light gray), 1E N381K R384L A385D (NRA) (black), 1E N381K R384L A385D D388T K389Q (NRADK) (white), 1E K389E (dark gray), and 1E R378E (hatched) from left to right as measured in 10 mM Ba2 .
Of the nine nonconserved residues, six positions involving significant changes in charge and/or size, as compared with the same sequence in 1C, were more thoroughly studied: R378E, N381K, R384L, A385D, D388T, K389Q, and K389E.
Y467S
protein
substitution
P15381
true positive
This distinction appears confirmed by a recent study that showed that mutations of the conserved Y (Tyr) residue in 1C (Y467S) had no significant effect on -subunit-induced modulation of whole-cell currents (Gerster et al., 1999).
R378K
protein
substitution
true positive
Q15878
R378K behaved like the wild-type 1E whereas R378Q displayed intermediate inactivation kinetics.
Biophysical Journal 80(1) 215228 AID Mutations in Voltage-Dependent Inactivation TABLE 1 Biophysical properties of 1E (CaV2.3) and 1C (CaV1.2) channels and mutants Inactivation (5 s) E0.5 (mV) 64 68 64 60 60 63 51 51 52 46 44 31 23 20 3 (9) 2 (7) 3 (4) 2 (3) 3 (5) 2 (5) 2 (6)* 2 (8)* 2 (10)* 3 (11) 2 (10) 2 (12) 3 (8) 4 (12) 3.5 2.8 3.1 2.8 2.2 3.2 3.3 3.1 2.9 2.7 2.3 4.2 3.4 3.1 z 0.4 0.5 0.5 0.6 0.7 0.3 0.2 0.2 0.4 0.3 0.5 0.2 0.4 0.4 E0.5 (mV) 18 15 11 15 14 14 21 12 19 16 14 14 13 11 2 (9) 2 (7) 2 (4) 1 (3) 4 (9) 3 (5) 5 (6) 3 (8) 4 (11) 3 (9) 3 (14) 4 (8) 5 (8) 3 (7) 6.1 6.0 5.7 5.8 5.4 4.8 5.1 4.8 6.1 6.2 5.5 6.7 9 10 Activation z 0.4 0.5 0.4 0.2 0.2 0.3 0.3 0.3 0.2 0.5 0.3 0.5 2 2 219 Channels expressed with 2b / 3 Peak IBa ( A) 3.7 1.9 3.6 2.4 4.1 2.7 2.6 3.1 3.8 3.5 4.6 2.0 3.3 3.9 1.3 (21) 0.4 (7) 1.4 (4) 1.2 (5) 1.1 (15) 1.5 (7) 0.6 (7) 0.5 (8) 1.0 (11) 0.7 (14) 1.7 (14) 0.4 (12) 0.3 (9) 0.5 (17) 1E wt 1E NRA-KLD NRAK-KLDQ NRADK-KLDTQ 1E K389E 1E R378K 1E R378A 1E R378Q 1E R378G 1E R378D 1E R378E 1C E462R 1C (XhoI) 1C wt Biophysical parameters of 1E and 1C wild-type (wt) and mutant channels expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
4 shows the family of wholecell Ba2 current traces of mutants R378K, R378Q, R378A, R378G, R378D, and R378E after expression in Xenopus oocytes.
Mutant 1E R378K displayed the fastest inactivation kinetics closely followed by R378A and R378Q.
At 10 mV, the rate of inactivation ranked as follows (from R e fastest to the slowest) 1E wt th R378K R378A 378Q R378G R378D R378E as seen by the r300 analysis shown in Fig.
The rate of R378K inactivation was comparable to 1E at 10 and 0 mV but differed slightly at higher membrane potential as its inactivation kinetics remained relatively insensitive to depolarization.
For R378Q, the r300 ratio was significantly (p 0.05) different from R378K at 10 mV, but these differences were attenuated at 20 mV.
The r300 ratios for the neutral residue R378A were noticeably similar to R378K and to the wild-type 1E channel at all membrane potentials.
R378A, a small and neutral residue, behaved like R378K, but R378G, which is also a small and neutral residue, behaved like negatively charged mutants R378D and R378E.
Furthermore, the activation potentials E0.5 for mutants R378E, R378D, R378G, R378A, and R378K were comparable to the E0.5 for the wild-type 1E channel.
Mutations at position 378 affect voltagedependent inactivation of the human 1E (CaV2.3) The next series of experiments was undertaken to evaluate whether the charge of the side-chain at position 378 played AID Mutations in Voltage-Dependent Inactivation 221 FIGURE 4 Mutants 1E R378K, 1E R378Q, and 1E R378A (A) and 1E R378G, 1E R378D, and 1E R378E (B) were expressed in Xenopus oocytes in the presence of 2b and 3 subunits.
Typical current traces are shown for the wild-type 1E and mutants R378K, R378Q, and R378E with an example of the voltage protocol used.
The inactivation data for 1E R378K superimposed quite closely with the inactivation data points for the wild-type 1E (Table 1).
The mean r300 ratios (the fraction of the whole-cell current remaining at the end of a 300-ms pulse) are shown SEM at four voltages from 10 to 20 mV for 1E wt (black), 1E R378K (light gray), 1E R378A (dark gray), 1E R378Q (white), 1E R378G (gray), 1E R378D (hatched), and 1E R378E (cross-hatched) from left to right.
At 10 mV, the r300 ratios for 1E R378K and 1E R378A were not significantly 0ifferent from 1E wt whereas those for 1E R378Q were different at p d .05 and those for 1E R378G, 1E R378D, and 1E R378E were significantly different at p 0.002.
The r300 ratios went from 0.04 0.01 0t 10 mV and 0.05 0.02 at 20 mV (n 7) for R378K, from 0.05 a .01 at 10 mV to 0.06 0.02 at 20 mV (n 4) for R378A, from 0.13 0.03 at 10 mV to 0.08 0.02 at 20 mV (n 7) for R378Q, from 0.27 0.03 at 10 mV and 0.21 0.01 at 20 mV (n 7) for R378G, from 0.25 0.03 at 10 mV to 0.19 0.02 at 20 mV (n 14) for R378D, and from 0.28 0.03 at 10 mV to 0.23 0.03 at 20 mV (n 14) for R378E as compared with 0.05 0.02 at 10 mV and 0.02 0 .01 at 20 mV (n 17) for 1E.
The activation potentials were comparable for all mutants although they increased slightly from 1E R378A 1E R378G 1E wt 1E R378K 1E R378D 1E R378E 1E R378Q.
Typical current traces for 1E wt, 1E R378K, 1E R378Q, and 1E R378E are shown from left to right.
The estimated mid-potentials of inactivation (E0.5) were comparable for 1E wt (f) and for 1E R378K ( ) whereas the mid-potentials of inactivation were shifted to the right for 1E R378A (*); 1E R378G (,), 1E R378Q (OE); 1E R378D ( ), and 1E R378E ().
Experiments were undertaken to investigate the role of electrostatic interaction in the inactivation kinetics of 1E with additional mutants R378K (positive), R378A (nonpolar and neutral), R378G (polar but Biophysical Journal 80(1) 215228 neutral), R378Q (polar but neutral), and R378D (negative).
At first glance, an electrostatic interaction appears to play a determinant role in the inactivation properties, as 1E R378K was the only mutant to reproduce the wild-type inactivation kinetics and voltage dependence.
Mutant R378A inactivated like R378K between 10 mV and 20 mV.
R378Q displayed intermediary inactivation kinetics at 10 mV but tended to inactivate like R378K at 20 mV.
Hence, there was no simple correlation between the inactivation kinetics of R378 mutants (R378E, R378D, R378K, R378A, R378Q, and R378G) and any single physicochemical property (charge, polarity, hydropathicity, and hydrophilicity).
10585923
full text
R615C
protein
substitution
true positive
P16960
Arg615-to-Cys615
Grammar mutation
12732620
full text
G61A
protein
substitution
true negative
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
On the other hand, three mutations (G61A, C72G, and G164 ins) induced exon skipping.
Q414X
protein
substitution
true positive
P13569
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
We have evaluated the possibility that some of the changes in the splicing pattern induced by the natural substitutions in exon 9, particularly the Q414X, might be related to nonsense mediated altered splicing.
On the other hand, in the F1 and F2 minigenes, C31T creates a stop codon (Q414X), whereas C155A does not.
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
Q456P
protein
substitution
true negative
The natural mutations Q456P, A455E, and V456F correspond to A146C, C155A and G157T, respectively.
V456F
protein
substitution
true positive
P13569
The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%).
Identification of Regulatory Elements of Splicing in CFTR Exon 9 --Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides.
On the contrary, the 153C and the 157T (V456F) variants did not significantly affect the splicing pattern.
The natural mutations Q456P, A455E, and V456F correspond to A146C, C155A and G157T, respectively.
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
C155A
protein
substitution
true negative
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
It is interesting to note that three nearby mutations, A146C, C155A, and G157T, at the 3 portion of the exon have a completely different effect on splicing.
We then tested, in the three different contexts, two of the natural substitutions with a splicing-inhibitory effect, the C31T and the C155A.
In the hCF context, the C31T and the C155A do not introduce a new stop codon but reduce the 65% hCF exon inclusion to 48 and 16%, respectively (Fig.
2D, compare hCF with hCF-C31T and hCF-C155A).
On the other hand, in the F1 and F2 minigenes, C31T creates a stop codon (Q414X), whereas C155A does not.
In fact, the 80% of exon inclusion in F1 and F2 is reduced in F1-C31T and F2-C31T to about 65%, and in F1-C155A and F2- C155A it is reduced to about 35% (Fig.
Identification of Regulatory Elements of Splicing in CFTR Exon 9 --Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides.
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
The natural mutations Q456P, A455E, and V456F correspond to A146C, C155A and G157T, respectively.
C155G
protein
substitution
true negative
UV-cross-linking experiments were done on mutants at position 155, on the 146C enhancing variant, on the 148A and 148G mutants, and on the double mutants 146C155G and 146C-155T (Fig.
G157T
protein
substitution
true negative
The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%).
It is interesting to note that three nearby mutations, A146C, C155A, and G157T, at the 3 portion of the exon have a completely different effect on splicing.
Identification of Regulatory Elements of Splicing in CFTR Exon 9 --Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides.
The natural mutations Q456P, A455E, and V456F correspond to A146C, C155A and G157T, respectively.
A20G
protein
substitution
true negative
In the F2 construct, the exon 9 was also placed in an open reading frame by the elimination of a stop codon at the 5 -end of the exon (A20G, TGA- TGG), deleting the nucleotide in position 23 (A23 ), and inserting a G at position 164 (G164 ).
The open reading frame in both of the mRNAs produced by these minigenes was restored by a base deletion at the 5 -end (F1 contains the T16 deletion, and F2 contains an A23 deletion along with an A20G substitution) and a G insertion at the 3 -end (G164 ) (for details, see "Experimental Procedures").
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
In particular, three nucleotide changes between positions 16 and 20 (T16 , T18G, A20G) induce exon inclusion, indicating the presence of a putative silencer element at the 5 portion of the exon, thus explaining the increase in exon inclusion observed in the F1 and F2 minigenes.
A146G
protein
substitution
true negative
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
A146C
protein
substitution
true negative
The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%).
It is interesting to note that three nearby mutations, A146C, C155A, and G157T, at the 3 portion of the exon have a completely different effect on splicing.
Identification of Regulatory Elements of Splicing in CFTR Exon 9 --Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides.
The natural mutations Q456P, A455E, and V456F correspond to A146C, C155A and G157T, respectively.
G147C
protein
substitution
true negative
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
T148G
protein
substitution
true negative
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
G424S
protein
substitution
true positive
P13569
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
T148C
protein
substitution
true negative
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
N414S
protein
substitution
true negative
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
I444S
protein
substitution
true positive
P13569
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
C31T
protein
substitution
true negative
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
We then tested, in the three different contexts, two of the natural substitutions with a splicing-inhibitory effect, the C31T and the C155A.
In the hCF context, the C31T and the C155A do not introduce a new stop codon but reduce the 65% hCF exon inclusion to 48 and 16%, respectively (Fig.
2D, compare hCF with hCF-C31T and hCF-C155A).
On the other hand, in the F1 and F2 minigenes, C31T creates a stop codon (Q414X), whereas C155A does not.
In fact, the 80% of exon inclusion in F1 and F2 is reduced in F1-C31T and F2-C31T to about 65%, and in F1-C155A and F2- C155A it is reduced to about 35% (Fig.
A455E
protein
substitution
true positive
P13569
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
Identification of Regulatory Elements of Splicing in CFTR Exon 9 --Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides.
The natural mutations Q456P, A455E, and V456F correspond to A146C, C155A and G157T, respectively.
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
For example, it appears that A455E can achieve adequate levels of chloride conduction at the cell surface (46, 47), causing only a partial CFTR protein processing defect (48).
Furthermore, the modulation by the concentration of splicing factors, which have an inhibitory effect on the CFTR exon 9 (26, 49) and a specific and possibly individual variation distribution, can provide an explanation for the phenotypic and tissue-specific variability in CF patients, particularly in those carrying the A455E substitution.
C72G
protein
substitution
true negative
On the other hand, three mutations (G61A, C72G, and G164 ins) induced exon skipping.
G118T
protein
substitution
true negative
The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%).
T18G
protein
substitution
true negative
In particular, three nucleotide changes between positions 16 and 20 (T16 , T18G, A20G) induce exon inclusion, indicating the presence of a putative silencer element at the 5 portion of the exon, thus explaining the increase in exon inclusion observed in the F1 and F2 minigenes.
Q452P
protein
substitution
true positive
P13569
The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%).
Identification of Regulatory Elements of Splicing in CFTR Exon 9 --Three natural missense mutations with completely different effects on splicing (Q452P (A146C), which induces exon inclusion; A455E (C155A), causing exon exclusion; and V456F (G157T), with no effect) are located within 15 nucleotides.
Extension of the mutagenesis in the 5 direction, including the Q452P (146C) variant showed that mutants from position 145 to 149, with the notable exception of the 148G, induced exon inclusion (Fig.
The 146C natural missense substitution (Q452P) with 95% of exon inclusion was analyzed in association with the nearby exon-skipping mutations in position 154 (C or T) and 155 (G or T).
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
T122G
protein
substitution
true negative
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
D443Y
protein
substitution
true positive
P13569
The G118T (D443Y) and G157T (V456F) mutations did not significantly affect the splicing pattern, whereas the A146C (Q452P) caused an almost complete inclusion on the exon (96%).
R protein matrices above thresholds WT sequence position AA change Nucleotide mutants Exon 9 Disruption of preexisting sites S SC35 New sites created by the mutations SR40 SF2 SR55 % WT A 15 T 16 T 18 G 19 A 20 C 31 A 43 A 44 46t49t G 61 66g67a69g C 72 G 118 120g122a123g T 122 A 144 C 145 A 146 C G A G T G G A C G A T A G G T G A G T C T C G C A C A C A C C T T C A G T T C T ins Q414X N414S G424S D443Y I444S Q452P G 147 T 148 T 149 G 150 T 151 G 153 G 154 C 155 G 156 G 157 G 164 V456F A455E 65 65 96 95 52 80 50 62 59 67 31 58 68 18 63 68 65 96 40 55 40 85 87 92 94 96 97 97 98 26 90 93 82 50 62 65 67 65 42 18 20 15 3 5 10 40 65 14 3.38 (13) SR40 0.28 (41) SR40 1.43 (41) SR401.01 (66) 3.21 (63) 2.24 (64) 2.20 (67) 2.01 (69) 2.24 (118) 3.02 (146) 3.23 (143) 3.46 (143) 2.81 (142) 2.70 (142) 2.49 (143) 2.99 (144) 3.38 (148) 3.00 (146) 3.15 (148) 3.00 (144) 2.99 (142) 2.66 (141) 3.03 (141) 2.53 (143) 4.05 (143) 2.47 (145) 3.46 (145) 3.53 (145) 2.76 (153) 1.98 (152) 3.59 (153) 3.82 (153) abundant splicing factors.
A4555E
protein
substitution
true negative
to the base affected by A4555E, to either A (the natural mutation), G, or T induced exon skipping (Fig.
G154C
protein
substitution
true negative
The site-directed mutants analyzed include five substitutions that create a new SF2/ASF high score motif (A146G, G147C, T148C, T148G, and C155A) and two nucleotide substitutions that do not create new SF2/ASF sites (A20G and G154C) (Table I).
In the absence of any cotransfected splicing factor and in comparison with normal exon 9, the mutations A20G, A146G, G147C, and T148C increase the percentage of exon inclusion, whereas T148G, G154C, and C155A induce exon skipping (Fig.
N418S
protein
substitution
true positive
P13569
Four of the natural substitutions, C31T (Q414X), G61A (G424S), T122G (I444S), and C155A (A455E), significantly decreased exon 9 inclusion to 48, 30, 40, and 16%, respectively, whereas only a modest decrease was evident for N418S.
10989292
full text
L1014F
protein
substitution
true negative
(1997) found no differences between para/TipE and para/TipE L1014F (kdr) expressed in Xenopus oocytes, with the V1/2 for activation= 16.9 mV vs 15.1 mV and the V1/2 for inactivation= 34 mV vs 31 mV.
(2000) found no differences between para/TipE, para/TipE L1014F (kdr) and para/TipE L10114F+M918T (super-kdr) expressed in Xenopus oocytes; activation V1/2= 13 to 17 mV and inactivation V1/2= 38 to 44 mV.
A1422V
protein
substitution
P35500
true positive
Second, the paraDN7 fly line has a Ala to Val (A1422V) change in the intracellular loop between S4 and S5 of the III domain, only a single amino acid position away from the anal- 0965-1748/00/$ - see front matter 2000 Elsevier Science Ltd.
/ Insect Biochemistry and Molecular Biology 30 (2000) 10511059 M1536I kdr para74 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 super kdr paraDN7 A1422V Fig.
A1422V in paraDN7 and M1536I in para74 (numbering is according to GenBank accession number AAB59195).
The paraDN7 line has a Ala to Val change (A1422V) in the intracellular loop between S4 and S5 of the III domain, a single amino acid position away from the analogous position in domain II of superkdr.
M1536I
protein
substitution
P35500
true positive
First, the para74 mutant fly line has a mutation analogous to that of kdr, but in the third domain, with a Met to Ile (M1536I) change within ten amino acid positions of the analogous position that kdr occupies in the second domain.
/ Insect Biochemistry and Molecular Biology 30 (2000) 10511059 M1536I kdr para74 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 super kdr paraDN7 A1422V Fig.
A1422V in paraDN7 and M1536I in para74 (numbering is according to GenBank accession number AAB59195).
Similar to kdr, the amino acid substitution (M1536I) associated with para74 lies within the S6 transmembrane region, but unlike kdr, the mutation is found in domain III instead of domain II.
M918T
protein
substitution
true negative
(2000) found no differences between para/TipE, para/TipE L1014F (kdr) and para/TipE L10114F+M918T (super-kdr) expressed in Xenopus oocytes; activation V1/2= 13 to 17 mV and inactivation V1/2= 38 to 44 mV.
9592081
full text
9539800
full text
C3S
protein
substitution
true positive
Q8VGC3
Formatting difference, Cys3,4Ser
C4S
protein
substitution
true negative
Q8VGC3
true positive
Upon elimination of palmitoylation by mut ating Cys-3 and Cys-4 to Ser, the inhibitor y ef fect was somewhat reduced F I G.
Formatting difference, Cys3,4Ser
9341133
full text
R615C
protein
substitution
true positive
P16960
26965 26966 Dantrolene Inhibition of Ryanodine Receptors EXPERIMENTAL PROCEDURES Materials--Pigs homozygous for either the MHS or normal RYR1 allele were obtained from the University of Minnesota Experimental Farm and genotyped on the basis of the Arg615 3 Cys MHS mutation (10).
This simple model of RYR1 activation during E-C coupling highlights the opposing effects of dantrolene and the MHS Arg615 3 Cys mutation on RYR1 activation by CaM and Ca2 and relates these effects to effects on the voltage dependence of RYR1 activation mediated via the transverse tubule voltage sensor (4, 35, 45).
This suggests that dantrolene may inhibit RYR1 channels via a mechanism that selectively counteracts the functional consequences of the MHS Arg615 3 Cys mutation.
Conversely, the MHS Arg615 3 Cys mutation is postulated to increase the stability of the sensitive (Closeds) state, thereby reproducing the observed increase in the fraction of MHS channels that may be activated by CaM and Ca2 (Fig.
In this way, the model reproduces the opposing effects of dantrolene and the Arg615 3 Cys mutation on the voltage dependence of activation that have been documented in studies using intact muscle fibers (4, 35, 37, 38).
In addition, it will be important to compare the functional consequences of other human MHS RYR1 mutations with those of the Arg615 3 Cys mutation (44), as the model predicts that rather than altering particular ligand binding sites, MH mutations may affect a global conformational transition in the RYR1 channel that is controlled by transverse tubule depolarization and results in increased channel activation by CaM and Ca2 .
12107168
full text
H532N
protein
substitution
true negative
The following primers were used in site-directed mutagenesis: P500T (mutation 1), 5 -GAACACTGCATACGTTTGATCCA;H532N (mutation 2), 5 -CCATTAATCCCAACCTGGGACCCT; F550I (mutation 3), 5 -CATCAAGTTCATCTTCATCTACA; and S867F (mutation 4), 5 -TACTTTCGCCTTCGATCCCATTGG, where the target bases are underlined.
S867F
protein
substitution
true positive
P19334
The following primers were used in site-directed mutagenesis: P500T (mutation 1), 5 -GAACACTGCATACGTTTGATCCA;H532N (mutation 2), 5 -CCATTAATCCCAACCTGGGACCCT; F550I (mutation 3), 5 -CATCAAGTTCATCTTCATCTACA; and S867F (mutation 4), 5 -TACTTTCGCCTTCGATCCCATTGG, where the target bases are underlined.
(9) showed that the TrpP365 mutant carries four protein sequence-altering mutations within its trp gene, P500T, H531N, F550I, and S867F.
H531N
protein
substitution
true positive
P19334
(9) showed that the TrpP365 mutant carries four protein sequence-altering mutations within its trp gene, P500T, H531N, F550I, and S867F.
P500T
protein
substitution
true positive
P19334
The following primers were used in site-directed mutagenesis: P500T (mutation 1), 5 -GAACACTGCATACGTTTGATCCA;H532N (mutation 2), 5 -CCATTAATCCCAACCTGGGACCCT; F550I (mutation 3), 5 -CATCAAGTTCATCTTCATCTACA; and S867F (mutation 4), 5 -TACTTTCGCCTTCGATCCCATTGG, where the target bases are underlined.
(9) showed that the TrpP365 mutant carries four protein sequence-altering mutations within its trp gene, P500T, H531N, F550I, and S867F.
F550I
protein
substitution
true positive
P19334
We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of TrpP365.
We now show that the above phenotypes are because of a single amino acid change, Phe-550 to Ile, in the fifth transmembrane segment of the TRP channel.
9726934
full text
Y152V
protein
substitution
P48544
true positive
K C hannel Pore Region Asymmetry 1337 FIGURE 9 GIRK4 channels with a nonconservative Y152V mutation (4V) are nonselective among Na , K , and Cs ; are constitutively activated; and are permeable to Ca2 .
12459180
full text
E3778F
protein
substitution
true positive
Q92736
Image mutation
N2386I
protein
substitution
true positive
Q92736
The histogram on the right illustrates dye intensities of colonies containing RyR1wt, RyR2wt, and the three mutagenized RyR2s: RyR2-ARVD2a (N2386I) [6], RyR2-ARVD2b (Y2392C) [9], and RyR2-VTSIP (R2474S) [7].
In vitro site-directed mutagenesis Site-directed mutagenesis was performed on RyR2-pYESTrp2, in order to reproduce three naturally occurring mutations: N2386I (RyR2-ARVD2a) [6], Y2392C (RyR2-ARVD2b) [9], and R2474S (RyR2-VTSIP) [7].
Moreover, we analysed and compared the interaction between FKBP12.6 and three mutagenized RyR2FBRs: RyR2ARVD2a (N2386I) [6], RyR2-ARVD2b (Y2392C) [9], and RyR2-VTSIP (R2474S) [7], bearing the naturally occurring mutations.
Although t he t wo ARVD 2 mutations roughly halved (N2386I) a nd almost ab ol ished (Y2392C) the staining reaction in a reproducible manner, the clinical phenotype of the corresponding ARVD2 families did not differ significantly.
N2386L
protein
substitution
true positive
Q92736
Image mutation
T2206R
protein
substitution
true positive
P21817
Image mutation
T2206M
protein
substitution
true positive
P21817
Image mutation
T2504M
protein
substitution
true positive
Q92736
Image mutation
R2163C
protein
substitution
true positive
P21817
Image mutation
R420W
protein
substitution
true positive
Q92736
Image mutation
I403M
protein
substitution
true positive
P21817
Image mutation
G248R
protein
substitution
true positive
P21817
Image mutation
R2163H
protein
substitution
true positive
P21817
Image mutation
R176Q
protein
substitution
true positive
Q92736
Image mutation
E4950K
protein
substitution
true positive
Q92736
Image mutation
A4860G
protein
substitution
true positive
Q92736
Image mutation
R2454C
protein
substitution
true positive
P21817
Image mutation
R2474S
protein
substitution
true positive
Q92736
The histogram on the right illustrates dye intensities of colonies containing RyR1wt, RyR2wt, and the three mutagenized RyR2s: RyR2-ARVD2a (N2386I) [6], RyR2-ARVD2b (Y2392C) [9], and RyR2-VTSIP (R2474S) [7].
In vitro site-directed mutagenesis Site-directed mutagenesis was performed on RyR2-pYESTrp2, in order to reproduce three naturally occurring mutations: N2386I (RyR2-ARVD2a) [6], Y2392C (RyR2-ARVD2b) [9], and R2474S (RyR2-VTSIP) [7].
Moreover, we analysed and compared the interaction between FKBP12.6 and three mutagenized RyR2FBRs: RyR2ARVD2a (N2386I) [6], RyR2-ARVD2b (Y2392C) [9], and RyR2-VTSIP (R2474S) [7], bearing the naturally occurring mutations.
As suggested by the staining intensity of the yeast colonies, the VTSI P mutation (R2474S) markedl y increased while the ARVD 2 mutations signi ficantl y dec re as ed the affi ni ty of Ry R2 FBR bindi ng to FKBP12.6.
R163C
protein
substitution
true positive
P21817
Image mutation
G341R
protein
substitution
true positive
P21817
Image mutation
R2458H
protein
substitution
true positive
P21817
Image mutation
R552S
protein
substitution
true positive
P21817
Image mutation
C35R
protein
substitution
true positive
P21817
Image mutation
R2458C
protein
substitution
true positive
P21817
Image mutation
R4201R
protein
substitution
true positive
Q92736
Image mutation
G3946S
protein
substitution
true positive
Q92736
Image mutation
E2311D
protein
substitution
true positive
Q92736
Image mutation
G2434R
protein
substitution
true positive
P21817
Image mutation
S2246L
protein
substitution
true positive
Q92736
Image mutation
N4104R
protein
substitution
true positive
Q92736
Image mutation
R2435H
protein
substitution
true positive
P21817
Image mutation
R2435L
protein
substitution
true positive
P21817
Image mutation
Y2392C
protein
substitution
true positive
Q92736
The histogram on the right illustrates dye intensities of colonies containing RyR1wt, RyR2wt, and the three mutagenized RyR2s: RyR2-ARVD2a (N2386I) [6], RyR2-ARVD2b (Y2392C) [9], and RyR2-VTSIP (R2474S) [7].
In vitro site-directed mutagenesis Site-directed mutagenesis was performed on RyR2-pYESTrp2, in order to reproduce three naturally occurring mutations: N2386I (RyR2-ARVD2a) [6], Y2392C (RyR2-ARVD2b) [9], and R2474S (RyR2-VTSIP) [7].
Moreover, we analysed and compared the interaction between FKBP12.6 and three mutagenized RyR2FBRs: RyR2ARVD2a (N2386I) [6], RyR2-ARVD2b (Y2392C) [9], and RyR2-VTSIP (R2474S) [7], bearing the naturally occurring mutations.
Although t he t wo ARVD 2 mutations roughly halved (N2386I) a nd almost ab ol ished (Y2392C) the staining reaction in a reproducible manner, the clinical phenotype of the corresponding ARVD2 families did not differ significantly.
P2328S
protein
substitution
true positive
Q92736
Image mutation
R4497C
protein
substitution
true positive
Q92736
Image mutation
I4867M
protein
substitution
true positive
Q92736
Image mutation
K506B
protein
substitution
true negative
The FKBP12.6 full-length coding sequence (GenBank HUMFK506B) was PCR-amplified from human heart cDNA with the following primers, containing XhoI and EcoRI sites: FKBP12.6-F: CTGGAATTCATGGGCGTGGAGATCGAG FKBP12.6-R: GACTCGAGTCACTCTAAGTTGAGCAGCTC The fragment was subcloned into pHybLex/Zeo (Invitrogen) and sequenced to confirm it was in-frame with LexA.
R614C
protein
substitution
true positive
P21817
Image mutation
V4653F
protein
substitution
true positive
Q92736
Image mutation
Y522S
protein
substitution
true positive
P21817
Image mutation
N4895D
protein
substitution
true positive
Q92736
Image mutation
L433P
protein
substitution
true positive
Q92736
Image mutation
R614L
protein
substitution
true positive
P21817
Image mutation
11564488
full text
C1N
protein
substitution
true negative
Examination between attacks showed cerebellar eye signs (gaze-evoked nystagmus and impaired suppression of the vestibulo-ocular reflex) and University Department of Clinical Neurology, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK (A Jouvenceau PhD, L H Eunson BSc, A Spauschus MD, Prof D M Kullmann FRCP, M G Hanna MD); INSERM U 549, 75014 Paris, France (A Jouvenceau); Department of Neurology, University of Bonn, Bonn, Germany (A Spauschus); Department of Paediatric Neurology, Newcastle General Hospital, Newcastle-upon-Tyne, UK (V Ramesh FRCP); and Fraser of Allander Neurosciences Unit, Royal Hospital for Sick Children, Glasgow, UK (S M Zuberi MRCP) Correspondence to: Prof Dimitri M Kullmann (e-mail: d.kullmann@ion.ucl.ac.uk) THE LANCET Vol 358 September 8, 2001 801 For personal use.
R1820STOP
protein
substitution
true negative
This mutation introduces a premature stop codon (R1820stop) resulting in complete loss of the C terminal region of the pore-forming subunit of this Ca2+ channel.
.1 20s Kv 1 Kv 18 /R .1 1 Kv A: selected traces from currents recorded from oocytes injected with wild-type (wt), mutant (R1820stop), and both wt and mutant 1A cDNA (Co-inj), evoked by 400 ms step depolarisation to 5 mV from a holding potential of 90 mV.
B: currents recorded in cells injected with wt were compared with cells injected with mutant R1820stop and with cells co-injected with wt: mutant (ratio 1:1), together with the auxiliary subunits 4 and 2 .
Figure 2: R1820stop mutant 1A is non-functional but exerts a dominant negative effect on wild type allele THE LANCET Vol 358 September 8, 2001 803 For personal use.
The R1820stop mutation lies between the last transmembrane segment (IVS6) and the intracellular C terminus (figure 1).
To investigate the possible consequences of the R1820stop mutation on ion channel function, we expressed wild-type and mutant 1A Ca2+ channel subunits in the presence of 4 and 2 subunits.
By contrast, oocytes injected with the R1820stop mutant cDNA showed only very small currents (22 nA [6]; n=22), which were indistinguishable from those obtained with uninjected control oocytes (15 nA [5]; n=9).
We confirmed that substituting subunits altered the kinetics as described in previously published research.15 In both cases, however, the current amplitude obtained by the coexpression of A Normalised conductance 1 08 wt coexpressed B 6 5 activation (msec) 40 4 3 2 1 0 06 04 02 0 80 60 40 20 Vtest (mV) 0 20 30 20 10 0 10 Vtest (mV) 20 30 40 C 12 Normalised conductance D 17 15 deactivation (msec) 120 80 Vhold 40 (mV) 0 40 13 11 09 07 05 160 140 120 Vtail (mV) 100 80 1 08 06 04 02 0 02 Figure 3: Comparison of wild-type (wt) and wt:R1820stop currents with respect to voltage and time-dependent kinetics A: voltage dependence of peak conductance of wt 1A alone and wt and mutant 1A cDNA co-expressed.
A possible explanation for the presence of overt epilepsy in association with the R1820stop mutation is that this mutation leads to a more substantial reduction in Ca2+ channel function than mutations associated with typical EA2.
10455017
full text
11812585
full text
R482STOP
protein
substitution
true positive
O00555
We also screened 210 cases for one of the two reported mutations in CACNB4, (the C1444T mutation at exon 13, resulting in an R482stop), with a similar assay using AluI, again with negative results.
R1820STOP
protein
substitution
true negative
The proband was heterozygous for an exon 36 mutation (C5733T), which introduces a stop codon (R1820stop) resulting in loss of the C-terminal region of the pore-forming 1A (Cav2.1) subunit.
12743166
full text
M1510I
protein
substitution
true negative
Note the different time scale for M1510I.
(C) Average time dependency of the inhibition of currents through 1G and the GC C5 and M1510I mutants by 3 (empty symbols) or 10 (filled symbols) M AA.
(E) Time constant of the inhibition of currents through 1G (black bars) and the GC C5 (white bars) and M1510I (dashed bar) mutants.
To diversify the structural determinants of fast inactivation to be tested for AA sensitivity, we also studied the mutant M1510I, which shows an 10-fold decrease in the rate of macroscopic inactivation with respect to 1G.
9 B shows examples of the inhibition of currents through 1G and the slow-inactivating mutants GC C5, G C1, and M1510I by 3 M AA.
The values of EC50 and Hill coefficient (N) for GC C5, G C1, and M1510I were significantly lower than for the wild type (Fig.
Sequence different than Swiss-Prot
9765517
full text
Y1771A
protein
substitution
P04775
true positive
Point mutation of comparable residues in NaIIa channels to alanine (NaIIa-F1764A, N1769A, and Y1771A) markedly altered receptor affinity for etidocaine (Ragsdale et al., 1994).
Y1586K
protein
substitution
P15390
true positive
Compared with 1, resting benzocaine block did not change at F1579K, decreased at N1584K, and increased at Y1586K.
Data are presented as mean standard error nels closely resembled the activation of 1 channels, whereas the midpoint voltages (V0.5) of activation for F1579K and Y1586K were 11 mV and 12 mV, respectively, more positive than the V0.5 value of activation for 1.
The V0.5 value of steady state inactivation for Y1586K channels was similar to that of 1 channels, whereas V0.5 value of steady state inactivation for F1579K and N1584K channels were 6 mV and 12 mV, respectively, more positive than that of 1.
For Y1586K (n 7), V0.5 and k values were 18.4 0.7 mV ( p 0.05 compared with 1) and 10.9 0.9 c mV ( p 0.05 compared with 1), respectively.
The average V0.5 values (50% availability) and k values for the fitted Boltzmann functions were: 79.5 2.1 mV and 6.2 0.4 mV, respectively for 1 (n 7); 73.6 0.8 mV ( p 0.05 compared with 1) and 5.5 0.1 mV ( 1p 0.04 compared with 1), respectively, for F1579K (n 11); 67.2 .2 mV ( p 0.05 compared with 1) and 7.1 0.4 mV, respectively, for N1584K (n 14); and 78.8 1.9 mV and 7.3 0.2 mV ( p 0.05 compared with 1), respectively, for Y1586K (n 7).
After a conditioning pulse to 140 mV, 50 M cocaine blocked similar percentages of current at resting 1 and Y1586K channels but blocked a smaller percentage of current at resting F1579K and N1584K channels.
Although the conditioning pulse to 70 mV in control saline elicited large amounts of slow inactivation at Y1586K channels (Fig.
2A, dashed line), the cocaine-induced reduction in test-current amplitude was much less at Y1586K channels than at 1 channels.
In contrast, conditioning pulses more positive than 120 mV elicited increasing amounts of slow inactivation at Y1586K channels.
Cocaine block of the resting channels ( 160 mV to 120 mV) was weak, although both F1579K and N1584K channels seemed less sensitive than either 1 or Y1586K.
Cocaine block of Y1586K channels resembled the block of 1 channels up to 100 mV, and conditioning pulses to between 90 mV and 70 mV produced modest increases in the block of Y1586K channels.
In B and C, n 6 ( 1), 6 (F1579K), 5 (N1584K) and 6 (Y1586K).
The decrease in cocaine block of Y1586K channels at 60 mV most likely resulted from modest amounts of channel activation and knockout of the drug by external Na ions (Wang, 1988).
B, The inactivated channel affinity (KI) of 1, F1579K, and Y1586K channels was determined by delivering a 10-sec conditioning pulse to 70 mV, followed by a 100-msec interval at 140 mV and a test pulse to 30 mV; for N1584K channels, a 10-sec conditioning pulse to 60 mV was used.
Cocaine had a similar affinity for resting 1 and Y1586K channels with KR values of 222.5 15.7 M (n 0 4) and 226.4 8.6 M (n 6; p .05), respectively.
We used a conditioning pulse to 70 mV for determining the inactivated channel affinities of 1, F1579K, and Y1586K.
In contrast, the KR/KI ratios at F1579K, N1584K, and Y1586K channels were 1.8, 1.9, and 3.1, respectively.
The reduction in inactivated channel affinity was smallest at Y1586K channels ( 6-fold) and was largest at N1584K channels ( 27-fold).
Compared with block of 1 channels, 1 mM benzocaine blocked a significantly smaller ( p 0.05) percentage of current at N1584K channels (35%) but a significantly larger ( p 0.05) percentage of current at Y1586K channels (55%).
Channel KR KI KR/KI 140 mV; KI values for KR/KR( 1, F1579K, and Y1586K were KI/KI( 1) 1) M 1 F1579K N1584K Y1586K 222.5 455.5 631.1 226.4 15.7 (4) 23.4* (4) 33.1* (7) 8.6 (6) 12.2 260.1 334.5 74.3 0.5 (5) 16.5* (4) 23.5* (7) 4.2* (4) 18.2 1.8 1.9 3.1 -- 2.1 2.8 1.0 -- 21.3 27.4 6.1 Numbers in parentheses indicate the number of cells; *, p 0.05 compared with 1.
Benzocaine shifted the h curve of 1 channels by 26 mV in the negative direction, whereas the negative shifts at F1579K, N1584K, and Y1586K channels were 7 mV, 4 mV, and 18 mV, respectively.
For both cocaine and benzocaine, the affinity of inactivated N1584K channels was most reduced and the affinity of inactivated Y1586K channels was least reduced.
Furthermore, the increase in benzocaine affinity at Y1586K can be explained if a cation- electron interaction between the aromatic ring on benzocaine and the amine Fig.
B, Averaged percentages of resting channels blocked by 1 mM benzocaine [n 10 ( 1), 9 (F1579K), 8 (N1584K) and 8 (Y1586K)].
The mean slope factors (k) of the fitted Boltzmann function were (control k value; benzocaine k value): 1 (6.2 mV 0.4; 8.9 0.3 mV*), F1579K (5.5 0.2 mV; 5.8 0.1 0 V), N1584K (6.9 0.4 mV; 7.0 0.6 mV), Y1586K (7.3 0.2 mV; 9.5 m .3 mV*), where * indicates a significant ( p 0.05) increase in the slope factor in the presence of benzocaine.
moiety on Y1586K is stronger than the hydrophobic interaction between the aromatic ring on benzocaine and the aromatic ring on tyrosine at Y1586.
Note that although approximately 10% of the N1584K channels did not fast inactivate, this small component could not be responsible for the cocaine resistance of the inactivated state in N1584K channels because both F1579K and Y1586K were cocaine-resistant, even though their steady state inactivation reached completion.
N1584K
protein
substitution
P15390
true positive
Lysine mutation at Y1586 did not alter resting channel affinity for cocaine but did reduce resting affinity at F1579K and N1584K by 2- and 3-fold, respectively.
Compared with 1, resting benzocaine block did not change at F1579K, decreased at N1584K, and increased at Y1586K.
The V0.5 value of steady state inactivation for Y1586K channels was similar to that of 1 channels, whereas V0.5 value of steady state inactivation for F1579K and N1584K channels were 6 mV and 12 mV, respectively, more positive than that of 1.
In addition, approximately 10% of the N1584K channels did not inactivate after conditioning pulses ranging from 50 mV to 35 mV.
Activation of N1584K chan- Fig.
For N1584K (n 8), V0.5 and k values were 28.0 1.6 mV and 7.3 0.5 mV ( p 0 .04 compared with 1), respectively.
The average V0.5 values (50% availability) and k values for the fitted Boltzmann functions were: 79.5 2.1 mV and 6.2 0.4 mV, respectively for 1 (n 7); 73.6 0.8 mV ( p 0.05 compared with 1) and 5.5 0.1 mV ( 1p 0.04 compared with 1), respectively, for F1579K (n 11); 67.2 .2 mV ( p 0.05 compared with 1) and 7.1 0.4 mV, respectively, for N1584K (n 14); and 78.8 1.9 mV and 7.3 0.2 mV ( p 0.05 compared with 1), respectively, for Y1586K (n 7).
After a conditioning pulse to 140 mV, 50 M cocaine blocked similar percentages of current at resting 1 and Y1586K channels but blocked a smaller percentage of current at resting F1579K and N1584K channels.
In control saline, 10-sec conditioning pulses more negative than 80 mV had little effect on the test current amplitude of 1, F1579K, or N1584K channels.
Conditioning pulses more positive than 80 mV, which began to elicit slow inactivation, had similar effects on test current amplitude at 1 and F1579K channels but had little effect on the current amplitude at N1584K channels.
Cocaine block of the resting channels ( 160 mV to 120 mV) was weak, although both F1579K and N1584K channels seemed less sensitive than either 1 or Y1586K.
In contrast, even the most positive conditioning pulses induced very little block of inactivated F1579K and N1584K channels.
In B and C, n 6 ( 1), 6 (F1579K), 5 (N1584K) and 6 (Y1586K).
B, The inactivated channel affinity (KI) of 1, F1579K, and Y1586K channels was determined by delivering a 10-sec conditioning pulse to 70 mV, followed by a 100-msec interval at 140 mV and a test pulse to 30 mV; for N1584K channels, a 10-sec conditioning pulse to 60 mV was used.
Compared with 1, resting F1579K and N1584K channels had a 2- to 3-fold ( p 0.05) lower affinity 23.4 M (n 4) and for cocaine with KR values of 455.5 631.1 33.1 M (n 7), respectively (Table 1).
Because the V0.5 value of steady state inactivation for N1584K channels was about 10 mV more positive than the V0.5 values for the other channels (Fig.
1B), we determined the inactivated channel affinity of N1584K channels using a conditioning pulse to 60 mV.
In contrast, the KR/KI ratios at F1579K, N1584K, and Y1586K channels were 1.8, 1.9, and 3.1, respectively.
The reduction in inactivated channel affinity was smallest at Y1586K channels ( 6-fold) and was largest at N1584K channels ( 27-fold).
Note that inactivated F1579K channels had a similar affinity for cocaine, and inactivated N1584K channels had a weaker affinity for cocaine compared with 1 resting affinity at 140 mV.
Compared with block of 1 channels, 1 mM benzocaine blocked a significantly smaller ( p 0.05) percentage of current at N1584K channels (35%) but a significantly larger ( p 0.05) percentage of current at Y1586K channels (55%).
KR values for all channels were determined at determined at 70 mV and the KI value for N1584K was determined at 60 mV.
Channel KR KI KR/KI 140 mV; KI values for KR/KR( 1, F1579K, and Y1586K were KI/KI( 1) 1) M 1 F1579K N1584K Y1586K 222.5 455.5 631.1 226.4 15.7 (4) 23.4* (4) 33.1* (7) 8.6 (6) 12.2 260.1 334.5 74.3 0.5 (5) 16.5* (4) 23.5* (7) 4.2* (4) 18.2 1.8 1.9 3.1 -- 2.1 2.8 1.0 -- 21.3 27.4 6.1 Numbers in parentheses indicate the number of cells; *, p 0.05 compared with 1.
Benzocaine shifted the h curve of 1 channels by 26 mV in the negative direction, whereas the negative shifts at F1579K, N1584K, and Y1586K channels were 7 mV, 4 mV, and 18 mV, respectively.
For both cocaine and benzocaine, the affinity of inactivated N1584K channels was most reduced and the affinity of inactivated Y1586K channels was least reduced.
B, Averaged percentages of resting channels blocked by 1 mM benzocaine [n 10 ( 1), 9 (F1579K), 8 (N1584K) and 8 (Y1586K)].
The mean slope factors (k) of the fitted Boltzmann function were (control k value; benzocaine k value): 1 (6.2 mV 0.4; 8.9 0.3 mV*), F1579K (5.5 0.2 mV; 5.8 0.1 0 V), N1584K (6.9 0.4 mV; 7.0 0.6 mV), Y1586K (7.3 0.2 mV; 9.5 m .3 mV*), where * indicates a significant ( p 0.05) increase in the slope factor in the presence of benzocaine.
Indeed, mutation 1-N1584K reduced cocaine affinity at resting channels by 3-fold and reduced the inactivated affinity by almost 30-fold.
The decreases in cocaine affinity at 1-N1584K can not be attributed to changes in gating because we determined KR at 140 mV where steady state inactivation is completely removed, and we determined KI at 60 mV where steady state inactivation of 1-N1584K was comparable with that of 1 at 70 mV.
Note that although approximately 10% of the N1584K channels did not fast inactivate, this small component could not be responsible for the cocaine resistance of the inactivated state in N1584K channels because both F1579K and Y1586K were cocaine-resistant, even though their steady state inactivation reached completion.
F1764A
protein
substitution
P04775
true positive
Point mutation of comparable residues in NaIIa channels to alanine (NaIIa-F1764A, N1769A, and Y1771A) markedly altered receptor affinity for etidocaine (Ragsdale et al., 1994).
N1769A
protein
substitution
P04775
true positive
Point mutation of comparable residues in NaIIa channels to alanine (NaIIa-F1764A, N1769A, and Y1771A) markedly altered receptor affinity for etidocaine (Ragsdale et al., 1994).
(1994) attributed the increase in resting etidocaine affinity at mutant NaIIa-N1769A ( 1N1584) to indirect effects because the model has the residue facing away from the channel pore as shown in the helical wheel plot of the D4-S6 segment in Fig.
F1579K
protein
substitution
P15390
true positive
Lysine mutation at Y1586 did not alter resting channel affinity for cocaine but did reduce resting affinity at F1579K and N1584K by 2- and 3-fold, respectively.
Compared with 1, resting benzocaine block did not change at F1579K, decreased at N1584K, and increased at Y1586K.
Data are presented as mean standard error nels closely resembled the activation of 1 channels, whereas the midpoint voltages (V0.5) of activation for F1579K and Y1586K were 11 mV and 12 mV, respectively, more positive than the V0.5 value of activation for 1.
The V0.5 value of steady state inactivation for Y1586K channels was similar to that of 1 channels, whereas V0.5 value of steady state inactivation for F1579K and N1584K channels were 6 mV and 12 mV, respectively, more positive than that of 1.
For F1579K (n 7), V0.5 and k values were 19.7 0.7 mV ( p 0.05 compared with 1) and 7.8 0.6 mV, respectively.
The average V0.5 values (50% availability) and k values for the fitted Boltzmann functions were: 79.5 2.1 mV and 6.2 0.4 mV, respectively for 1 (n 7); 73.6 0.8 mV ( p 0.05 compared with 1) and 5.5 0.1 mV ( 1p 0.04 compared with 1), respectively, for F1579K (n 11); 67.2 .2 mV ( p 0.05 compared with 1) and 7.1 0.4 mV, respectively, for N1584K (n 14); and 78.8 1.9 mV and 7.3 0.2 mV ( p 0.05 compared with 1), respectively, for Y1586K (n 7).
After a conditioning pulse to 140 mV, 50 M cocaine blocked similar percentages of current at resting 1 and Y1586K channels but blocked a smaller percentage of current at resting F1579K and N1584K channels.
In control saline, 10-sec conditioning pulses more negative than 80 mV had little effect on the test current amplitude of 1, F1579K, or N1584K channels.
Conditioning pulses more positive than 80 mV, which began to elicit slow inactivation, had similar effects on test current amplitude at 1 and F1579K channels but had little effect on the current amplitude at N1584K channels.
Cocaine block of the resting channels ( 160 mV to 120 mV) was weak, although both F1579K and N1584K channels seemed less sensitive than either 1 or Y1586K.
In contrast, even the most positive conditioning pulses induced very little block of inactivated F1579K and N1584K channels.
In B and C, n 6 ( 1), 6 (F1579K), 5 (N1584K) and 6 (Y1586K).
B, The inactivated channel affinity (KI) of 1, F1579K, and Y1586K channels was determined by delivering a 10-sec conditioning pulse to 70 mV, followed by a 100-msec interval at 140 mV and a test pulse to 30 mV; for N1584K channels, a 10-sec conditioning pulse to 60 mV was used.
Compared with 1, resting F1579K and N1584K channels had a 2- to 3-fold ( p 0.05) lower affinity 23.4 M (n 4) and for cocaine with KR values of 455.5 631.1 33.1 M (n 7), respectively (Table 1).
We used a conditioning pulse to 70 mV for determining the inactivated channel affinities of 1, F1579K, and Y1586K.
In contrast, the KR/KI ratios at F1579K, N1584K, and Y1586K channels were 1.8, 1.9, and 3.1, respectively.
Note that inactivated F1579K channels had a similar affinity for cocaine, and inactivated N1584K channels had a weaker affinity for cocaine compared with 1 resting affinity at 140 mV.
On average, 1 mM benzocaine blocked a similar amount of current at 1 channels (40%) and F1579K channels (41%; p 0.05).
Channel KR KI KR/KI 140 mV; KI values for KR/KR( 1, F1579K, and Y1586K were KI/KI( 1) 1) M 1 F1579K N1584K Y1586K 222.5 455.5 631.1 226.4 15.7 (4) 23.4* (4) 33.1* (7) 8.6 (6) 12.2 260.1 334.5 74.3 0.5 (5) 16.5* (4) 23.5* (7) 4.2* (4) 18.2 1.8 1.9 3.1 -- 2.1 2.8 1.0 -- 21.3 27.4 6.1 Numbers in parentheses indicate the number of cells; *, p 0.05 compared with 1.
Benzocaine shifted the h curve of 1 channels by 26 mV in the negative direction, whereas the negative shifts at F1579K, N1584K, and Y1586K channels were 7 mV, 4 mV, and 18 mV, respectively.
B, Averaged percentages of resting channels blocked by 1 mM benzocaine [n 10 ( 1), 9 (F1579K), 8 (N1584K) and 8 (Y1586K)].
The mean slope factors (k) of the fitted Boltzmann function were (control k value; benzocaine k value): 1 (6.2 mV 0.4; 8.9 0.3 mV*), F1579K (5.5 0.2 mV; 5.8 0.1 0 V), N1584K (6.9 0.4 mV; 7.0 0.6 mV), Y1586K (7.3 0.2 mV; 9.5 m .3 mV*), where * indicates a significant ( p 0.05) increase in the slope factor in the presence of benzocaine.
Note that although approximately 10% of the N1584K channels did not fast inactivate, this small component could not be responsible for the cocaine resistance of the inactivated state in N1584K channels because both F1579K and Y1586K were cocaine-resistant, even though their steady state inactivation reached completion.
The reduction in inactivated channel binding of benzocaine at F1579K may be caused by charge interference or steric interaction between the amino group of lysine and the amino group of benzocaine when the channel occupies the high affinity conformation.
10050000
full text
V6T
protein
substitution
true positive
P22460
K7L
protein
substitution
true negative
I369L
protein
substitution
true negative
1993) where inactivation is ascribed to a single I369L change, and can be modulated in the deep pore region (Kiss & Korn, 1998).
R487V
protein
substitution
true positive
P22460
1993), whereas the positively charged residue K gives a rapid Properties of the point mutants R487Y and R487V C_type inactivation (Schlief et al.
5 (R487V), the process of slow inactivation was again unaffected (data not shown).
5 Inactivation and recovery from inactivation in the mutants R487Y and R487V In many K channels a regulatory site for external cations has been shown to lie in the outer pore mouth.
R487Y
protein
substitution
true positive
P22460
Inactivation in the mutant channel R487Y TEA was added to the bath just prior to the recording of trace 1.
B, normalized inactivating R487Y current in control and during exposure to 1 m or 25 m TEACl in the external bath.
C, currents from cell transfected with R487Y recorded during two-pulse steady-state inactivation protocol.
1993), whereas the positively charged residue K gives a rapid Properties of the point mutants R487Y and R487V C_type inactivation (Schlief et al.
9A, control currents are shown from R487Y-hKv1.
A and B, original data from WT and R487Y channels.
m., n = 8) in WT and R487Y, respectively.
Recovery from inactivation in WT and R487Y 326 D.
9C and the mean steady-state inactivation relations for R487Y are shown in Fig.
Not only was the onset of slow inactivation unaffected in the R487Y mutant compared with the WT channel, but recovery was unaffected as well (Fig.
10A and B, for WT and the R487Y mutant, respectively.
Mean data on recovery from inactivation in WT and R487Y channels are shown in Fig.
No difference was observed between WT and R487Y channels.
Data in both cases have been fitted to a single exponential recovery function with similar time constants of 1107 and 976 ms in WT and R487Y, respectively.
5 Inactivation and recovery from inactivation in the mutants R487Y and R487V In many K channels a regulatory site for external cations has been shown to lie in the outer pore mouth.
We observed that the mutation R487Y did confer high TEA sensitivity to the normally TEAinsensitive hKv1.
The rate of inactivation in TEA-blocked channels was not appreciably slowed, and the inactivation kinetics of unblocked R487Y channels were not significantly different from WT channels.
W434F
protein
substitution
true negative
How does the W434F mutation block current in Shaker potassium channels.
10049317
full text
R13Q
protein
substitution
true negative
This inactivation process showed similar features to C-type inactivation in Shaker K channels because it was suppressed by the presence of an extracellular pore-blocking ligand, the mutant -conotoxin GIIIA ( -CTX), R13Q.
-CTX R13Q was used because it only partially blocks single channel current (Becker et al., 1992), thus allowing channel gating mechanisms to be studied when all channels are occupied by toxin (French et al., 1996).
The time course of recovery of normalized peak inward currents was best fit with the double exponential function by air oxidation, -CTX R13Q was purified to near-homogeneity by HPLC.
The -CTX analog R13Q was synthesized by solid state synthesis on a polystyrene-based Rink amide resin on an Applied Biosystems 431A synthesizer.
Modulation of ultra-slow inactivation by the mutant -conotoxin GIIIA, R13Q Studies involving site-directed mutagenesis have demonstrated that fast inactivation is mediated by the interaction of a cytoplasmic loop between the third and the fourth domains and some part of the inner vestibule (Stuhmer et al., 1989; West et al., 1992; McPhee et al., 1994; Smith and Goldin, 1997).
Furthermore, a mutant of -CTX, R13Q, shows residual current of 2530% of the control when the toxin is bound to the channel (French et al., 1996), allowing assessment of channel gating kinetics in the toxin-bound state.
The Kd of -CTX R13Q binding to the mutant K1237E is 10 M (unpublished data).
3 shows the effect of a near-saturating ( 3x Kd) concentration of 27 M -CTX R13Q on the time course of recovery from inactivation by the mutant channel K1237E.
Superfusion of 27 M -CTX R13Q was started, reducing peak inward current by 75 80%.
After recovery from ultra-slow inactivation was complete, perfusion with a near-saturating concentration of 27 M -CTX R13Q was started (arrow, "27 M R13Q").
After the reduction of peak Na current by -CTX R13Q had reached a steady-state level, which reflects the residual current flowing through partially blocked channels, the inactivation/recovery protocol was repeated.
Binding of -CTX R13Q reduced the amount of ultra-slow recovery from inactivation.
1), was 0.81 0.05 under control conditions, and 0.49 0.09 during perfusion with 27 M -CTX R13Q (p 0.01), while 2 was reduced from 112.6 1 6.8 s at control to 66.3 13.6 s with -CTX R13Q (p 0.05).
4, -CTX R13Q significantly slowed the development of ultra-slow inactivation.
If residence of -CTX R13Q in the channel mouth reduces the probability of the channel being in ultra-slow inactivated state, then the toxin-induced acceleration of recovery from ultra-slow inactivation should be concentration-dependent.
This apparent concentration-dependence suggests that the effect of -CTX R13Q on ultra-slow inactivation is dependent on occupation by R13Q of its blocking site on the channel.
The tight inverse correlation between the fraction of channels that are ultra-slow inactivated and the estimated fraction of channels bound by R13Q (Fig.
-CTX R13Q has been shown to shift voltage-dependent channel activation gating, perhaps by an electrostatic effect on the voltage sensor (French et al., 1996).
It is, however, absence of -CTX R13Q, recovery was not completed until several hundred seconds following the return to the holding potential.
During superfusion with -CTX R13Q, recovery from ultra-slow inactivation had a much faster time course.
The data points were normalized to the final level of ultra-slow recovery and the time courses of ultra-slow recovery with and without superfusion with -CTX R13Q were fitted with Eq.
Binding of -CTX R13Q markedly reduced the amplitude of ultra-slow recovery from inactivation (A2) by 30%, from 0.81 0.05 under control conditions to 0.49 0.09 during perfusion with 27 M -CTX R13Q 1p ( 0.01, n 6, Fig.
2 was reduced from 112.6 06.8 s at control to 66.3 13.6 s with -CTX R13Q (p .05).
If -CTX R13Q directly alters the time course of recovery from ultra-slow inactivation, the toxin should also affect the rate of development of this inactivated state.
During superfusion with 27 M -CTX R13Q the K1237E channels were depolarized from 120 mV to 30 mV for variable lengths of time, and the time course of recovery at 120 mV was monitored for each prepulse duration by applying brief, repetitive (20-ms) test pulses to 10 mV at FIGURE 4 -CTX R13Q slows entry into the ultra-slow inactivated state.
During superfusion with 27 M -CTX R13Q, the membrane potential of oocytes expressing K1237E channels was depolarized from 120 mV to 30 mV for variable lengths of time, and the time course of recovery at 120 mV was monitored for each prepulse duration as described in the legend to Fig.
Hence -CTX R13Q prolonged entry into the ultraslow inactivated state consistent with a direct effect of -CTX R13Q on ultra-slow inactivation.
1340 Biophysical Journal Volume 76 March 1999 FIGURE 5 The effect of -CTX R13Q on ultra-slow inactivation in the mutant K1237E is dependent on the toxin concentration.
(A) Recovery from ultra-slow inactivation was determined during a toxin-free control, during superfusion with 10 M -CTX R13Q, and, repeatedly, during wash-out of -CTX R13Q.
After recovery from ultra-slow inactivation was complete, superfusion with 10 M -CTX R13Q was started (arrow, "start R13Q").
Recovery from ultra-slow inactivation was substantially accelerated by -CTX R13Q ("R13Q", top).
The time course of recovery from ultra-slow inactivation is substantially accelerated by -CTX R13Q.
Superfusion with -CTX R13Q resulted in a decrease of both peak inward current (i.e., toxin block), and in a reduction of the amplitude of ultra-slow recovery from inactivation (i.e., of the number of channels entering the ultra-slow inactivated state).
Hence, the magnitude of the effect of -CTX R13Q on ultra-slow inactivation is correlated with a measure of the toxin remaining in the bath.
(D) Inverse correlation between the amplitude of ultra-slow inactivation, and the estimated fraction of channels bound by R13Q, fbound, from the experiment shown in (A).
The occupancy of the channel by -CTX R13Q was calculated from the current reduction, assuming that 70% of the single channel current is blocked (French et al., 1996.: fbound 1 {[(I/Icontrol) 0.3]/0.7}.
Parenthetically, we note that if block of the single channel current for the channel mutant K1237E were all-or-none, rather than only 70% complete, the predicted probability of ultra-slow inactivation for this protocol with all channels R13Q-bound would be decreased slightly to 0.15.
Ultra-Slow Inactivation in Na C hannels 1341 FIGURE 6 The reduction of ultra-slow inactivation by -CTX R13Q does not appear to be produced by an electrostatic effect on the voltage sensor.
(A) The reduction in the relative amplitude of ultra-slow inactivation by -CTX R13Q is independent of the prepulse voltage.
Recovery at 120 mV from a 300-s prepulse to the indicated potentials was monitored in K1237E during a drug-free control, and during perfusion with 27 M -CTX R13Q.
The reduction of ultra-slow recovery from inactivation by -CTX R13Q was similar for prepulse potentials of 30 mV, 0 mV, and 30 mV.
Little change with -CTX R13Q was observed at more negative potentials ( 60 mV, 80 mV) where only small amounts of ultra-slow recovery were observed under control conditions (overlap of symbols for control with data points acquired during superfusion with -CTX R13Q).
Number of experiments (control/ -CTX R13Q): 30 mV (0/5), 0 mV (8/8), 30 mV (11/11), 60 mV (6/6), and 80 mV (6/6).
(B) The reduction of ultra-slow inactivation by -CTX R13Q in K1237E cannot be reproduced by changes in recovery potential.
The -CTX R13Q-induced reduction of ultra-slow inactivation at a recovery potential of 120 mV (R13Q, 27 M; same data as inset to Fig.
3, after -CTX R13Q) could not be reproduced by changing the recovery potential under drug-free conditions.
Thus, toxin-induced shifts in recovery potential are unlikely to account for the reduction in ultra-slow inactivation by -CTX R13Q.
Thus, a toxin-induced shift of the inactivation potential cannot account for the reduction in ultra-slow inactivation by -CTX R13Q.
By comparison, superfusion with -CTX R13Q reduced the number of channels recovering in an ultra-slow fashion at 120 mV much more than any change in recovery potential.
These results support the hypothesis that -CTX R13Q interferes with ultra-slow inactivation by means of a direct interaction with the outer vestibule.
The results argue against a strong voltage-dependence of -CTX R13Q binding under our experimental conditions.
Thus, the lack of voltage-dependence of the -CTX R13Q effect in our experiments appears to reflect the lower voltage sensitivity of the interaction of these blocking toxins with closed Na channels in the absence of batrachotoxin.
A very slow inactivation process has been described for squid giant axon Na channels (Adelman and Palti, 1969; Rudy, 1978), called "ultra-slow" inactivation by Fox (1976), and probably underlies the requirement for prolonged, strong hyperpolarization to observe voltage ac- 27 M -CTX R13Q.
Block of ultra-slow inactivation by -CTX R13Q Several molecules are known to block the Na channel by binding in the outer channel vestibule.
The toxin mutant -CTX R13Q has the interesting property that binding only partially occludes the channel, reducing but not abolishing single channel current (Becker et al., 1992; French et al., 1996; Chang et al., 1998).
5) suggested that -CTX R13Q was affecting only toxin bound channels.
The data are consistent with the idea that binding of -CTX R13Q to the channel mutant K1237E favors a conformation of the channel which, with respect to ultraslow inactivation, behaves very much like the wild-type channel in the absence of the peptide.
The reduction of ultra-slow inactivation by -CTX R13Q in K1237E may be analogous to the effects of TEA and K on C-type inactivation in Shaker K channels.
The mechanism of -CTX R13Q is unclear, however.
If -CTX R13Q affects a structural rearrangement, it is unlikely that the effect is directly on K1237.
Consequently, structural rearrangements prevented by -CTX R13Q are more likely to be produced by widespread interactions of the toxin with the outer vestibule of the channel.
Based upon the known interactions of the toxin with the channel, the modifying effect of -CTX R13Q is not likely to be by a direct interaction with the residue at position 1237, but rather at a more superficial site.
In this regard the modification of ultra-slow inactivation by -CTX R13Q in Na channels may be different from the effect of TEA on C-type inactivation in Shaker K channels.
-CTX R13Q may act as a "splint" in the outer vestibule, stabilizing the structure of this region of the channel.
Denis McMaster of the Peptide Synthesis Laboratory, University of Calgary Faculty of Medicine, for providing the peptide -CTX R13Q.
K1237R
protein
substitution
P15390
true positive
We refer to this process as "ultra-slow inactivation." By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within 60 s, with only 20 30% of the current showing ultra-slow recovery.
The mutations of the lysine residue at amino acid position 1237 to Arg (K1237R), Ser (K1237S), and Glu (K1237E) were made by three primer PCR (Bowman et al., 1990).
Peak Na currents of wild-type 1 and mutant K1237R channels recovered almost completely within 60 s.
In contrast to 1 and K1237R, channels carrying the mutations K1237S and K1237E did not completely recover until more than 300 s had elapsed.
2, the longer time constant of recovery, corresponded to a component representing 20% (A2 in Table 1) of the overall time course of recovery of 1 and K1237R channels.
Ultra-Slow Inactivation in Na C hannels c 1337 urrents after a K1237E 8.9 129.3 0.2 0.8 2.9 10.6 0.04* 0.08* TABLE 1 Parameters of recovery of Na 300-s prepulse to 30 mV 1 1 2 A1 A2 16.6 76.9 0.77 0.23 0.6 22.1 0.07 0.07 K1237R 11.2 74.6 0.79 0.21 2.3 13.7 0.07 0.03 K1237S 10.2 97.5 0.15 0.85 1.3 7.4 0.03* 0.03* Shown are time constants of recovery from slow inactivation ( 1, s) and ultra-slow inactivation ( 2, s) following a 300-s prepulse to 30 mV, as well as the individual amplitudes (A1, A2).
The faster component of recovery (A1 in Table 1) accounting for 80% of recovery of 1 and K1237R channels, and for only 1520% of recovery of K1237S and K1237E mutants, had a time constant of 10 s ( 1 in Table 1).
The mutations in domains III, in which the charge of the residue was altered, exhibited a substantial amount of slow recovery from inactivation, whereas wild-type 1, and the charge-conserving mutation in domain III, K1237R, recover much faster.
The latter time constant is similar to the time constant of the ultra-slow component of recovery in the wild-type 1 and in the charge preserving mutation K1237R (Table 1).
In contrast to the mutations K1237S and K1237E, the charge-preserving mutation K1237R did not increase the component of the ultra-slow inactivation process.
(1996) found that the charge-preserving mutation K1237R caused loss of selectivity between Na and K .
K1237S
protein
substitution
P15390
true positive
When skeletal muscle subunits ( 1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of 100 s.
The mutations of the lysine residue at amino acid position 1237 to Arg (K1237R), Ser (K1237S), and Glu (K1237E) were made by three primer PCR (Bowman et al., 1990).
In contrast to 1 and K1237R, channels carrying the mutations K1237S and K1237E did not completely recover until more than 300 s had elapsed.
Ultra-Slow Inactivation in Na C hannels c 1337 urrents after a K1237E 8.9 129.3 0.2 0.8 2.9 10.6 0.04* 0.08* TABLE 1 Parameters of recovery of Na 300-s prepulse to 30 mV 1 1 2 A1 A2 16.6 76.9 0.77 0.23 0.6 22.1 0.07 0.07 K1237R 11.2 74.6 0.79 0.21 2.3 13.7 0.07 0.03 K1237S 10.2 97.5 0.15 0.85 1.3 7.4 0.03* 0.03* Shown are time constants of recovery from slow inactivation ( 1, s) and ultra-slow inactivation ( 2, s) following a 300-s prepulse to 30 mV, as well as the individual amplitudes (A1, A2).
dramatically increased to 80% in channels carrying the mutations K1237S and K1237E.
More than 90% of the mutant K1237S and K1237E channels could be driven into the ultra-slow inactivated state by increasing the duration of the prepulse in our experiments to 600 s (Fig.
The faster component of recovery (A1 in Table 1) accounting for 80% of recovery of 1 and K1237R channels, and for only 1520% of recovery of K1237S and K1237E mutants, had a time constant of 10 s ( 1 in Table 1).
The mutations K1237S and K1237E, however, appear to favor entry into a state of ultra-slow inactivation.
The time constants of development of ultra-slow inactivation were 119.0 4.9 s, and 206.9 23.3 s for K1237S and K1237E, respectively.
Number of experiments for each duration of the depolarizing prepulse: K1237S: 5 s (2), 15 s (4), 30 s (6), 60 s (7), 90 s (4), 180 s (4), 300 s (7), 450 s (4), 600 s (7).
After a 600-s depolarization, 90 100% of the channels with the mutations K1237S or K1237E had entered an ultra-slow mode.
The time constants of develop4 ent of ultra-slow inactivation at 30 mV were 119.0 m .9 s and 206.9 23.3 s for K1237S and K1237E, respectively.
The similarity of the time constants of that process with the one we observed with the 1 K1237S and K1237E mutations led us to use Fox's terminology.
In contrast to the mutations K1237S and K1237E, the charge-preserving mutation K1237R did not increase the component of the ultra-slow inactivation process.
W402C
protein
substitution
P15390
true positive
Tomaselli and his colleagues reported that the mutation W402C eliminated slow inactivation in 1 Na channels (Tomaselli et al., 1995; Balser et al., 1996).
K1237E
protein
substitution
P15390
true positive
When skeletal muscle subunits ( 1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of 100 s.
Coexpression of the rat brain 1 subunit along with the K1237E subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation.
The mutations of the lysine residue at amino acid position 1237 to Arg (K1237R), Ser (K1237S), and Glu (K1237E) were made by three primer PCR (Bowman et al., 1990).
RESULTS The first indication of the presence of a very slow inactivation process was the occurrence of a progressive increase in peak Na current in Xenopus oocytes expressing the mutation K1237E.
After establishing a negative holding potential of 120 mV by voltage clamp, the peak Na current assessed by consecutive short test pulses increased to a stable level over a several minute period in K1237E, whereas in wild-type 1 a stable current level was reached within 60 s.
After the Na current had stabilized at a higher level, a subsequent prolonged depolarization was followed by a similar slow recovery in K1237E (Fig.
In contrast to 1 and K1237R, channels carrying the mutations K1237S and K1237E did not completely recover until more than 300 s had elapsed.
Ultra-Slow Inactivation in Na C hannels c 1337 urrents after a K1237E 8.9 129.3 0.2 0.8 2.9 10.6 0.04* 0.08* TABLE 1 Parameters of recovery of Na 300-s prepulse to 30 mV 1 1 2 A1 A2 16.6 76.9 0.77 0.23 0.6 22.1 0.07 0.07 K1237R 11.2 74.6 0.79 0.21 2.3 13.7 0.07 0.03 K1237S 10.2 97.5 0.15 0.85 1.3 7.4 0.03* 0.03* Shown are time constants of recovery from slow inactivation ( 1, s) and ultra-slow inactivation ( 2, s) following a 300-s prepulse to 30 mV, as well as the individual amplitudes (A1, A2).
dramatically increased to 80% in channels carrying the mutations K1237S and K1237E.
More than 90% of the mutant K1237S and K1237E channels could be driven into the ultra-slow inactivated state by increasing the duration of the prepulse in our experiments to 600 s (Fig.
The faster component of recovery (A1 in Table 1) accounting for 80% of recovery of 1 and K1237R channels, and for only 1520% of recovery of K1237S and K1237E mutants, had a time constant of 10 s ( 1 in Table 1).
The mutations K1237S and K1237E, however, appear to favor entry into a state of ultra-slow inactivation.
(A) Slowly increasing inward currents through K1237E channels elicited by test pulses after the holding potential was returned to 120 mV.
The time constants of development of ultra-slow inactivation were 119.0 4.9 s, and 206.9 23.3 s for K1237S and K1237E, respectively.
K1237E: 5 s (2), 15 s (7), 30 s (6), 60 s (7), 90 s (3), 180 s (5), 300 s (6), 450 s (4), 600 s (7).
After a 600-s depolarization, 90 100% of the channels with the mutations K1237S or K1237E had entered an ultra-slow mode.
The time constants of develop4 ent of ultra-slow inactivation at 30 mV were 119.0 m .9 s and 206.9 23.3 s for K1237S and K1237E, respectively.
We tested whether the 1 subunit had an effect on ultra-slow inactivation of the mutant K1237E channel.
Recovery from a long (300-s) prepulse to 30 mV was determined in the mutant K1237E (Fig.
The Kd of -CTX R13Q binding to the mutant K1237E is 10 M (unpublished data).
3 shows the effect of a near-saturating ( 3x Kd) concentration of 27 M -CTX R13Q on the time course of recovery from inactivation by the mutant channel K1237E.
During the control period, K1237E channels were depolarized to 30 mV for 300 s and recovery from ultra-slow inactivation was monitored by 20-ms depolarizations from 120 mV to 10 mV at 20-s intervals, as before.
1 was coexpressed with the subunit carrying the DIII K1237E mutation (n 3).
Ultra-Slow Inactivation in Na C hannels 1339 FIGURE 3 Recovery from ultra-slow inactivation in K1237E is modulated by a mutant -conotoxin known to bind at the outer vestibule.
During wash-out of toxin in K1237E channels, consecutive recovery curves from ultra-slow inactivation were determined.
During superfusion with 27 M -CTX R13Q the K1237E channels were depolarized from 120 mV to 30 mV for variable lengths of time, and the time course of recovery at 120 mV was monitored for each prepulse duration by applying brief, repetitive (20-ms) test pulses to 10 mV at FIGURE 4 -CTX R13Q slows entry into the ultra-slow inactivated state.
During superfusion with 27 M -CTX R13Q, the membrane potential of oocytes expressing K1237E channels was depolarized from 120 mV to 30 mV for variable lengths of time, and the time course of recovery at 120 mV was monitored for each prepulse duration as described in the legend to Fig.
1340 Biophysical Journal Volume 76 March 1999 FIGURE 5 The effect of -CTX R13Q on ultra-slow inactivation in the mutant K1237E is dependent on the toxin concentration.
Parenthetically, we note that if block of the single channel current for the channel mutant K1237E were all-or-none, rather than only 70% complete, the predicted probability of ultra-slow inactivation for this protocol with all channels R13Q-bound would be decreased slightly to 0.15.
If no peptide is bound, this amplitude is predicted to be between 0.9 and 1.0; which is in agreement with the amplitude of ultra-slow inactivation in K1237E, in the absence of toxin.
6 A shows the effect of variations in the potential of a 300 s inactivating prepulse on ultra-slow recovery from inactivation by the mutant channel K1237E.
Recovery at 120 mV from a 300-s prepulse to the indicated potentials was monitored in K1237E during a drug-free control, and during perfusion with 27 M -CTX R13Q.
(B) The reduction of ultra-slow inactivation by -CTX R13Q in K1237E cannot be reproduced by changes in recovery potential.
The similarity of the time constants of that process with the one we observed with the 1 K1237S and K1237E mutations led us to use Fox's terminology.
In contrast to the mutations K1237S and K1237E, the charge-preserving mutation K1237R did not increase the component of the ultra-slow inactivation process.
In the mutant K1237E, recovery from inactivation produced by a short (1-s) depolarizing prepulse also follows a doubleexponential time course, suggesting states analogous to both fast and slow inactivation in this mutant (Fig.
2, the amount of slow inactivation (but not ultra-slow inactivation) in the mutant K1237E is also substantially reduced by coexpression with the 1 subunit.
Ultra-Slow Inactivation in Na C hannels 1343 Na channels with the K1237E mutation we found that this molecule destabilized the ultra-slow inactivated state both by slowing entry into the state (Fig.
The data are consistent with the idea that binding of -CTX R13Q to the channel mutant K1237E favors a conformation of the channel which, with respect to ultraslow inactivation, behaves very much like the wild-type channel in the absence of the peptide.
The reduction of ultra-slow inactivation by -CTX R13Q in K1237E may be analogous to the effects of TEA and K on C-type inactivation in Shaker K channels.
9559222
full text
A3C
protein
substitution
true negative
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
R347P
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
Class IV mutations result in a reduction in the amount of chloride current (e.g., R117H, R347P, S1251N mutations) while class V mutations result in a reduction in the amount of a normally functioning CFTR protein (e.g., A455E, 3849 10kbC3 T mutations).
R553X
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
R1162X
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
G551D
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
In class III, the CFTR protein reaches the cell membrane but is irresponsive to cAMP stimulation (e.g., G551D, N1303K, G85E mutations).
W1282X
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
A455E
protein
substitution
true positive
P13569
Results: CF patients carrying the A455E mutation, usually associated with pancreatic sufficiency, had lower sweat chloride concentrations than those carrying mutations associated with pancreatic insufficiency ( F508 and 621 1G3 T).
of Patients 47 28 6 12 5 5 4 3 2 2 Mean Chloride Concentration (mEq/L) (SD) 102.77 (8.43) 103.5 (7.4) 94.17 (10.87) 77.08 (18.48) 100.6 (11.67) 112.4 (2.97) 106.5 (4.12) 102.33 (11.5) 71 89.5 Mean Chloride Concentration (mEq/L) (SD) 102.28 (8.56) 104.55 (8.14) 81.68 (18.14) Minimum Value (mEq/L) 77 87 74 54 87 108 102 91 62 84 Minimum Value (mEq/L) 77 84 54 Maximum Value (mEq/L) 119 120 106 107 117 116 110 114 80 95 Maximum Value (mEq/L) 119 120 107 Genotype F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 Y1092X/ F508 or 621 1G3T 621 1G3T/621 1G3T 621 1G3T/711 1G3T F508/other Other/other 621-other No.
of Patients 54 40 19 Mutation F508 621 1G3T A455E Molecular characterization has been performed on all identified living CF patients (12, De Braekeleer M.
Three CFTR mutations account for 93.7% of the CF chromosomes; these are the F508 (63.4%), 621 1G3 T (22.3%), and A455E (8%) mutations.
All the 114 CF patients in the present study had pancreatic insufficiency but 7 with a A455E/ F508 genotype, 2 with a A455E/621 1G3 T genotype and 1 with a R177C/A455E genotype who were pancreatic sufficient.
Patients with the A455E/ F508 or the 621 1G3 T/ A455E genotype had significantly lower chloride values than those homozygous for the F508 mutation (p 0.05).
A significant difference was also found between CF patients carrying the A455E mutation on one chromosome and the F508 or 621 1G3 T mutation on the other chromosome (p 0.05).
It was the result of significant differences between the A455E group and the other two groups, F508 and 621 1G3 T (p 0.05).
Patients were classified as hemizygous for the F508 mutation if the other chromosome did not carry the 621 1G3 T, nor the A455E mutation.
Patients carrying the A455E mutation on one chromosome were included in the A455E mutation category, independently of the second mutation.
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
However, the follow-up of CF patients with one mild allele (A455E) has shown that some PS patients may develop pancreatic insufficiency at a later stage of the disease (14).
Class IV mutations result in a reduction in the amount of chloride current (e.g., R117H, R347P, S1251N mutations) while class V mutations result in a reduction in the amount of a normally functioning CFTR protein (e.g., A455E, 3849 10kbC3 T mutations).
It was the case for F508/ F508 versus 621 1G3 T/621 1G3 T or A455E/ F508 versus A455E/621 1G3 T.
R334W
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
R177C
protein
substitution
true negative
All the 114 CF patients in the present study had pancreatic insufficiency but 7 with a A455E/ F508 genotype, 2 with a A455E/621 1G3 T genotype and 1 with a R177C/A455E genotype who were pancreatic sufficient.
Typo
G85E
protein
substitution
true positive
P13569
In class III, the CFTR protein reaches the cell membrane but is irresponsive to cAMP stimulation (e.g., G551D, N1303K, G85E mutations).
S1251N
protein
substitution
true positive
P13569
Class IV mutations result in a reduction in the amount of chloride current (e.g., R117H, R347P, S1251N mutations) while class V mutations result in a reduction in the amount of a normally functioning CFTR protein (e.g., A455E, 3849 10kbC3 T mutations).
Y1092X
protein
substitution
true positive
P13569
of Patients 47 28 6 12 5 5 4 3 2 2 Mean Chloride Concentration (mEq/L) (SD) 102.77 (8.43) 103.5 (7.4) 94.17 (10.87) 77.08 (18.48) 100.6 (11.67) 112.4 (2.97) 106.5 (4.12) 102.33 (11.5) 71 89.5 Mean Chloride Concentration (mEq/L) (SD) 102.28 (8.56) 104.55 (8.14) 81.68 (18.14) Minimum Value (mEq/L) 77 87 74 54 87 108 102 91 62 84 Minimum Value (mEq/L) 77 84 54 Maximum Value (mEq/L) 119 120 106 107 117 116 110 114 80 95 Maximum Value (mEq/L) 119 120 107 Genotype F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 Y1092X/ F508 or 621 1G3T 621 1G3T/621 1G3T 621 1G3T/711 1G3T F508/other Other/other 621-other No.
There were no significant differences in sweat chloride values between patients homozygous for the F508 mutation and those having a F508/ 621 1G3 T genotype or being hemizygous for the Y1092X or 711 1G3 T mutation (p 0.05).
In class I, there is no protein synthesis (e.g., Y1092X, 621 1G3 T, 711 1G3 T mutations).
R117H
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
Class IV mutations result in a reduction in the amount of chloride current (e.g., R117H, R347P, S1251N mutations) while class V mutations result in a reduction in the amount of a normally functioning CFTR protein (e.g., A455E, 3849 10kbC3 T mutations).
G542X
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
N1303K
protein
substitution
true positive
P13569
of CF Patients 128 46 56 13 26 22 20 328 6 9 5 10 17 7 16 22 58 9 4 47 28 6 12 Mean Chloride Concentration (mmol/L) (SD) 109 (23) 105 (18) 104 (24) 110 (18) 107 (36) 100 (20) 82 (19) 106 (22) 61 (11) 41 (12) 100 (26) 108 (19) 98 (12) 124 113 (12) 109 (11) 101 (16) 99 (13) 105 (20) 103 (8) 103 (7) 94 (11) 77 (18) Pancreatic Status Pl Pl Pl Pl Pl Pl PS Pl PS PS (6) Pl Pl (6) Pl Pl Pl Pl Pl Pl Pl Pl Pl Pl/PS Pl/PS Genotype G542X/ F508 R553X/ F508 N1303K/ F508 W1282X/ F508 1717-1G3A/ F508 621 1G3T/ F508 R117H/ F508 F508/ F508 3849 10kb C3T/ F508 3849 10kb C3T/ F508 R347P/ F508 R334W/ F508 1811 1.6kb A3C/ F508a 3905insT/ F508 W1282X/W1282X W1282X/ F508 G551D/ F508 R1162X/R1162X 1949del84/ F508 F508/ F508 621 1G3T/ F508 621 1G3T/A455E A455E/ F508 a References 18 18 18 18 18 18 18 18 19 20 21 22 23 24 25 25 26 27 28 This study This study This study This study Or other `severe' mutations.
In class III, the CFTR protein reaches the cell membrane but is irresponsive to cAMP stimulation (e.g., G551D, N1303K, G85E mutations).
11251047
full text
E244Q
protein
substitution
true negative
As no clear change of phenotype from WT could be observed in the E244Q mutant, the specificity of the effects of mutations at E299 and E224 was supported.
N171D
protein
substitution
true positive
P35560
The corresponding amino acid residues were negatively charged in all strong inward rectifiers and in Kir1.1, which also showed strong rectification when a negatively charged amino acid was introduced into the M2 region (N171D) (Lu & MacKinnon, 1994).
G231E
protein
substitution
true negative
The G231E mutation in WT sWIRK also did not clearly strengthen the inward rectification (data not shown).
E299Q
protein
substitution
true positive
P35561
It was additionally observed that E224GE299S and E224GE299Q had similar inward rectification properties, and that D172NE299S and D172NE299Q were also similar (Fig.
However, as there was a difference in the inward rectification properties between E299S and E299Q (Fig.
Phenotype of the D172NE224GE299S mutant and comparison with that of sWIRK To examine whether the three sites focused on in the p r e s e n t study determine the inward rectification property completely, two triple mutants of Kir2.1, D172NE224GE299S and D172NE224GE299Q, were made.
1996), E299Q of sWIRK showed a slight outward rectification, and no clear sign of inward rectification.
In contrast, a weak but clear inward rectification remained in the triple mutants of Kir2.1 (D172NE224GE299S and D172NE224GE299Q).
Macroscopic current recordings under two-electrode voltage clamp of the sWIRK mutant E179Q and two triple mutants of Kir2.1, D172NE224GE299S and D172NE224G E299Q, expressed in Xenopus oocytes A detailed explanation of AC is given in Fig.
Although this possibility remains in the present study, the relatively intact channel function of E299S and E299Q suggests that it is more straightforward to assume that the negative charges of E299 and E224 contribute to the permeation pathway.
E299R
protein
substitution
true positive
P35561
To examine further the effect of electrostatic charge at this residue, E299R and E299K mutants were also prepared.
E299S
protein
substitution
true positive
P35561
Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224GlyGlu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells.
(a) Glu299Ser showed a weaker inward rectification, a slower activation upon hyperpolarization, a slower decay of the outward current upon depolarization, a lower sensitivity to block by cytoplasmic spermine and a smaller singlechannel conductance than WT.
(b) The features of Glu224Gly were similar to those of Glu299Ser.
(c) In the double mutant (Glu224GlyGlu299Ser), the differences from WT described above were more prominent.
E224G
protein
substitution
true positive
P35561
Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224GlyGlu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells.
(b) The features of Glu224Gly were similar to those of Glu299Ser.
(c) In the double mutant (Glu224GlyGlu299Ser), the differences from WT described above were more prominent.
S311E
protein
substitution
true negative
It was also observed that the introduction of the S311E mutation to WT sWIRK (E179, G231, S311E) J.
531.3 Inward rectification property of Kir2.1 E299S mutant 659 or to the sWIRK E179Q mutant (E179Q, G231, S311E) did not significantly increase inward rectification (data not shown).
E179Q
protein
substitution
true negative
By mutating E179 of sWIRK to Gln (Q) (E179Q, G231), the inward rectification property was almost completely lost and it showed outward rectification even in the presence of cytoplasmic blockers (Kubo et al.
The E179Q mutant of sWIRK, which has no n e g a t i v e charges at the three sites in question (E179QG231S311) were also made.
Macroscopic current recordings under two-electrode voltage clamp of the sWIRK mutant E179Q and two triple mutants of Kir2.1, D172NE224GE299S and D172NE224G E299Q, expressed in Xenopus oocytes A detailed explanation of AC is given in Fig.
Other amino acid residues for the control of inward rectification There was a marked difference in the rectification property of E179Q of sWIRK (E179Q, G231, S311) and those of the triple mutants of Kir2.1 (D172N, E224G, E299S or Q) (Fig.
531.3 Inward rectification property of Kir2.1 E299S mutant 659 or to the sWIRK E179Q mutant (E179Q, G231, S311E) did not significantly increase inward rectification (data not shown).
E299K
protein
substitution
true positive
P35561
To examine further the effect of electrostatic charge at this residue, E299R and E299K mutants were also prepared.
D172N
protein
substitution
true positive
P35561
As this feature clearly differed from the double mutant of Kir2.1 (D172NE224G) (Yang et al.
RESULTS Macroscopic current recordings of WT and mutant Kir2.1 The electrophysiological properties of a point mutant of Kir2.1 in which Glu at position 299 was mutated to Ser (E299S) were compared with those of WT Kir2.1 and the D172N and E224G mutants (Fig.
The properties of the double mutants of the three sites, D172NE224G, D172NE299S and E224GE299S, were also analysed (Fig.
(1) The D172N mutation weakened the inward rectification slightly, and removed the activation phase upon hyperpolarization (Figs 2 and 3).
(2) Both the E224G and E299S mutations remarkably weakened the inward rectification of WT Kir2.1 and the D172N mutant.
Comparison of the inward rectification properties of WT and mutant Kir2.1 The intensity of the inward rectification of WT Kir2.1 and the D172N, E224G, E299S and E224GE299S mutants is compared in Fig.
It is clear that the D172N mutant showed slightly weaker inward rectification than WT, and both E224G and E299S mutants showed much less intense inward rectification.
It was additionally observed that E224GE299S and E224GE299Q had similar inward rectification properties, and that D172NE299S and D172NE299Q were also similar (Fig.
Macroscopic current recordings under two-electrode voltage clamp of WT Kir2.1 (IRK1) and D172N, E224G and E299S mutants expressed in Xenopus oocytes A, representative current recordings in 10 mM KG.
Comparison of the activation kinetics of WT and mutant Kir2.1 The activation phase upon hyperpolarization was not clearly observed in the D172N (Fig.
2A), D172NE224G or D172NE299S mutants (Fig.
Macroscopic current recordings under two-electrode voltage clamp of double mutants of Kir2.1 (D172NE224G, D172NE299S and E224GE299S) expressed in Xenopus oocytes A detailed explanation is given in Fig.
The symbols used are as follows: WT, 0; D172N, 9; E224G, ; E299S, 1; E224GE299S, 8.
Phenotype of the D172NE224GE299S mutant and comparison with that of sWIRK To examine whether the three sites focused on in the p r e s e n t study determine the inward rectification property completely, two triple mutants of Kir2.1, D172NE224GE299S and D172NE224GE299Q, were made.
In contrast, a weak but clear inward rectification remained in the triple mutants of Kir2.1 (D172NE224GE299S and D172NE224GE299Q).
Macroscopic current recordings under two-electrode voltage clamp of the sWIRK mutant E179Q and two triple mutants of Kir2.1, D172NE224GE299S and D172NE224G E299Q, expressed in Xenopus oocytes A detailed explanation of AC is given in Fig.
531.3 Differences between the functional role of D172 and those of E224 and E299 In the D172N mutant, the sensitivity to block by spermine was decreased and the activation phase at hyperpolarized potentials disappeared (Stanfield et al.
In contrast with D172N, the activation speed at hyperpolarized potentials (unblocking) and the decay speed of the outward current at depolarized potentials (blocking) were slower than those of WT (Figs 5 and 6).
Other amino acid residues for the control of inward rectification There was a marked difference in the rectification property of E179Q of sWIRK (E179Q, G231, S311) and those of the triple mutants of Kir2.1 (D172N, E224G, E299S or Q) (Fig.
12941782
full text
S1387F
protein
substitution
true negative
In this case, and in a second child we have identified with the Ser1387Phe mutation, surgery revealed diffuse disease, but no second mutation was found.
E1507K
protein
substitution
true negative
(8) described an extended Finnish family with congenital hyperinsulinism caused by a different dominantly expressed SUR1 missense mutation, Glu1507Lys.
The obligate carriers in Huopio et al.'s (8) dominant SUR1 Glu1507Lys family had histories consistent with symptomatic hypoglycemia in early childhood.
(34) reported decreased insulin responses to oral and intravenous glucose tolerance tests in the nondiabetic adult carriers of the Glu1507Lys mutation, which they interpret as evidence of progressive -cell failure.
Thus, the poor insulin responses to glucose noted in adults with the Glu1507Lys SUR1 mutation by Huopio et al.
This is similar to the studies of the dominant Glu1507Lys mutation reported by Huopio et al.
Preliminary electrophysiological studies of the delSer1387 show evidence of residual channel activity similar to that found with the Glu1507Lys mutation.
9391164
full text
Y1586A
protein
substitution
P15390
true positive
In c ontrast, the double mut ant E755A plus Y1586A showed sign ificantly reduced external block (10.1 1.1%, n 6) and E755A plus F1579A showed no detect able block (1.0 0.5%, n 5) during 11- to 12-min ex posure to external 500 M QX222 (P 0.001).
These c onclusions are derived f rom the f o l l o w i n g f i n d i n g s i n s e l e c t i v i t y f i l t e r m u t a n t s : (i ) a c c e l e r a t e d rec over y f rom internal QX block; (ii) a similar time c ourse of rec over y f rom QX block when QX was applied f rom the inside or the outside; (iii) no block or reduced block by external QX in double mut ant (E755A F1579A, E755A Y1586A); and (iv) outside QX ac cess impeded by the presence of outer vestibule 3.
E755A
protein
substitution
P15390
true positive
Mut ation of the other selectivit y residues, D400A (domain I), E755A (domain II), and A1529D (domain I V) allowed block by exter nally applied quater nar y memb r a n e - i m p e r m e a n t d e r i v a t i v es o f l i d o c a i n e ( Q X 3 1 4 a n d QX222) and ac celerated rec over y f r om block by inter nal QX314.
Block by outside QX314 in E755A was inhibited by mut ation of residues in t ransmembrane segment S6 of domain I V that are thought to be par t of an c ter nal binding site.
On the other hand, the domain II mut ant E755A increased af fin it y 2-fold (Kd 284 26 M) and the domain III mut ant K1237E increased af fin it y 4-fold (Kd 154 16 M) (P 0.001).
2 A), E755A (Fig.
On average, block was 20.5 2.5% (n 9), 38.2 2.5% (n 8), and 29.2 2.6% (n 5) for D400A, E755A, and A1529D, respectively, af ter 11 min of per fusion.
E755A and A1529D also showed 32.1 2.5% (n 10) and 60.8 3.0% (n 5) block 10 min af ter external application of 500 M QX314.
Peak currents 10 11 min af ter ex posure to indicated c oncentrations of lidocaine were nor malized to that in the c ontrol for 1 w ild-t ype (W T, E), D400A (domain I, F), E755A (domain II, OE), K1237E (domain III, ) and A1529D (domain I V, ) in A and for W T (E), K1237E ( ), K1237S (OE) and K1237R (F) in B.
Kd values are 572, 552, 284, 154, 281, 521, and 611 M for W T, D400A, E755A, K1237E, K1237S, K1237R, and A1529D, respectively.
( B ) E755A.
In c ontrast, the selectiv it y filter mut ants that per mitted block by external QX222, E755A, and A1529D showed much faster rec over y than that of w ild t ype (time c onst ants were 1,263 and 228 s, respectively).
In E755A, the time c onst ant of rec over y was 111 s when QX222 was applied internally and 116 s when applied externally (Fig.
Degrees of use-dependent block by internal QX314 were 87.5, 78.3, 65.6, and 70.9% for w ild t ype, D400A, E755A, and A1529D, respectively.
The smooth lines are single ex ponential fits, and time c onst ants are 1,264 s for E755A and 228 s for A1529D.
For internal application, 50 nl of 3 mM QX222 or QX314 was microinjected into ooc y tes ex pressing E755A (E) and A1529D (,), respectively.
For external application, 500 M QX222 or 500 M QX314 was added to the bath solution for E755A (F) and A1529D (OE), respectively and per fused until rec over y protoc ol was c omplete.
The smooth lines are single ex ponential fits for E755A (solid lines) and A1529D (dashed lines) dat a.
For E755A, time c onst ants are 111 s for internal and 116 s for external QX222.
The E755A channel mut ant has a ver y low tox in af fin it y, mak ing protection ex periments impossible.
The double mut ant c omposed of E755A and either the mut ation F1579 or Y1586 in domain I V, S6 was tested.
If F1579 or Y1586 is involved in local anesthetic binding site and E755A creates an ac cess pathway to the internal binding site f rom the outside, no block or reduced block by external QX would be ex pected in a double mut ant.
When 500 M QX222 was applied to the bath solution, E755A showed 34% block (Fig.
In c ontrast, the double mut ant E755A plus Y1586A showed sign ificantly reduced external block (10.1 1.1%, n 6) and E755A plus F1579A showed no detect able block (1.0 0.5%, n 5) during 11- to 12-min ex posure to external 500 M QX222 (P 0.001).
These findings demonstrate that mut ant E755A creates a sec ond ac cess pathway for QX222 to reach its internal binding site.
E755A increases K per meabilit y over Na (7) and A1529D is predicted to increase K per meabilit y f rom A1529E dat a (6).
Because K (radius 1.33 ) is bigger than 0.95 ), the selectiv it y filter region in E755A Na (radius and A1529D may have a larger diameter, and c onsequently, QX c ompounds might directly pass through it.
E755A, the negative charge reducing mut ation in domain II, increased lidocaine af fin it y, but D400A, a similar charge reducing mut ation of domain I, and A1529D, the negative residue replacement of domain I V, had no ef fect on af fin it y.
Increased af fin it y by E755A does not fit an electrost atic ef fect, and its mechan ism is unclear.
As mentioned below, it is possible that E755A mut ation increases the rested channel af fin it y for lidocaine by open ing a sec ond ac cess pathway to the site, but this is unlikely because lidocaine block was judged at steady st ate.
E755A is the only mut ant af fecting both dr ug ac cess and binding.
L oss of the attraction w ith E755A might improve binding by allow ing a better hydrophobic interaction and results in a paradox ical improvement in lidocaine af fin it y.
These c onclusions are derived f rom the f o l l o w i n g f i n d i n g s i n s e l e c t i v i t y f i l t e r m u t a n t s : (i ) a c c e l e r a t e d rec over y f rom internal QX block; (ii) a similar time c ourse of rec over y f rom QX block when QX was applied f rom the inside or the outside; (iii) no block or reduced block by external QX in double mut ant (E755A F1579A, E755A Y1586A); and (iv) outside QX ac cess impeded by the presence of outer vestibule 3.
K1237E
protein
substitution
P15390
true positive
Mut ation of the put ative domain III selectivit y f ilter residue of the adult rat skelet al muscle Na channel ( 1) K1237E increased resting lidocaine block, but no change was obser ved in block by neut ral analogs of lidocaine.
On the other hand, the domain II mut ant E755A increased af fin it y 2-fold (Kd 284 26 M) and the domain III mut ant K1237E increased af fin it y 4-fold (Kd 154 16 M) (P 0.001).
Because the larger af fin it y change was obser ved in K1237E, we further studied the ef fects of replacement of K1237 w ith other amino acids.
Ton ic block by phenol was not inf luenced by the mut ant K1237E (w ild-t ype Kd, 6.5 2.7 mM; K1237E Kd, 5.6 1.6 mM; P 0.203; Fig.
Consistent w ith this result, Kd values for ton ic block by benzocaine were similar bet ween w 82 M) and K1237E (440 0 ild-t ype (553 57 M; P .619) values.
Interestingly, even though K1237 interacted electrost atically w ith lidocaine, neither K1237E nor K1237S allowed external QX222 block (Fig.
2D; 6.6 1.2% block, n 5 for K1237E, and 4.3 1.4% block, n 4 for K1237S).
Peak currents 10 11 min af ter ex posure to indicated c oncentrations of lidocaine were nor malized to that in the c ontrol for 1 w ild-t ype (W T, E), D400A (domain I, F), E755A (domain II, OE), K1237E (domain III, ) and A1529D (domain I V, ) in A and for W T (E), K1237E ( ), K1237S (OE) and K1237R (F) in B.
Kd values are 572, 552, 284, 154, 281, 521, and 611 M for W T, D400A, E755A, K1237E, K1237S, K1237R, and A1529D, respectively.
( C ) Ef fects of K1237E on ton ic block by the hydrophobic lidocaine analogs phenol and benzocaine.
Peak currents 10 11 min af ter ex posure to the indicated c oncentrations of phenol (E, F) or benzocaine (,, OE) were nor malized to that in the c ontrol for W T (E, ,) and K1237E (F, OE) and plotted as a function of phenol or benzocaine c oncentrations.
For phenol block, Kd values are 6.5 and 5.6 mM for W T and K1237E, respectively.
For benzocaine block, Kd values are 553 and 440 M for W T and K1237E, respectively.
( D ) K1237E (E) and K1237S ( ).
Peak currents elicited by 35-ms pulses to 10 mV f rom a holding potential of 100, 110 (for A1529D), or 120 mV (for K1237E) at 20-s inter vals were c or malized to peak current in c ontrol and plotted as relative Na n urrents (relative INa).
The mut ation K1237E did shif t the availabilit y cur ve about 20 mV, requiring these ex periments to be made f rom a holding potential of 120 mV, where channels were fully available.
K1237E is per meable to Ba 2 (6), but K1237S seems to increase Ba2 block of Na currents because Na currents were dramatically reduced af ter changing to Ba 2 solution (dat a not shown).
However, K1237E also increases K per meabilit y (6) but shows no detect able block by external QX, and D400A allows external QX block w ithout an increase of K per meabilit y (7).
The Kd value for w ild t ype was 572 M and for the double-charge mut ant K1237E was 154 M.
The obser ved 3.7-fold increase in lidocaine binding af fin it y seen w ith K1237E requires an increase in binding energ y of 0.8 kcal mol.
D400A
protein
substitution
P15390
true positive
Mut ation of the other selectivit y residues, D400A (domain I), E755A (domain II), and A1529D (domain I V) allowed block by exter nally applied quater nar y memb r a n e - i m p e r m e a n t d e r i v a t i v es o f l i d o c a i n e ( Q X 3 1 4 a n d QX222) and ac celerated rec over y f r om block by inter nal QX314.
Neo-sax itox in and tet r odotox in, w hich oc clude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively.
In w ild t ype, the lidocaine dissociation c onst ant (Kd) was 572 38 M and no change was obser ved in domain I mut ant D400A (Kd 552 39 M) or domain I V mut ant A1529D (Kd 611 35 M) (Fig.
In c ontrast, D400A (Fig.
On average, block was 20.5 2.5% (n 9), 38.2 2.5% (n 8), and 29.2 2.6% (n 5) for D400A, E755A, and A1529D, respectively, af ter 11 min of per fusion.
Peak currents 10 11 min af ter ex posure to indicated c oncentrations of lidocaine were nor malized to that in the c ontrol for 1 w ild-t ype (W T, E), D400A (domain I, F), E755A (domain II, OE), K1237E (domain III, ) and A1529D (domain I V, ) in A and for W T (E), K1237E ( ), K1237S (OE) and K1237R (F) in B.
Kd values are 572, 552, 284, 154, 281, 521, and 611 M for W T, D400A, E755A, K1237E, K1237S, K1237R, and A1529D, respectively.
( A ) Wild t ype (W T, E) and D400A ( ).
D400A allowed moderate external block, and rec over y was marginally faster than that of w ild t ype.
Degrees of use-dependent block by internal QX314 were 87.5, 78.3, 65.6, and 70.9% for w ild t ype, D400A, E755A, and A1529D, respectively.
In D400A, 1 M neo-STX produced 100% block and 1 mM QX314 blocked 21% of the current af ter 12 min of per fusion (Fig.
USA 94 (1997) 14129 upon washout of the mixture than that of QX314 alone was c onfir med in three and six additional ooc y tes for D400A and A1529D, respectively.
The graphs show t ypical examples of ex periments in D400A ( A ) and A1529D ( B ) .
The bar shows the period during ex posure to 1 M neo-STX (n-STX), the mixture of 1 M n-STX plus 1 mM QX314 (n-STX QX314), or 1 mM QX314 in D400A ( A ) ; and 500 M QX314, the mixture of 5 M T TX plus 500 M QX314 (T TX QX314), or 5 M T TX in A1529D ( B ) .
However, K1237E also increases K per meabilit y (6) but shows no detect able block by external QX, and D400A allows external QX block w ithout an increase of K per meabilit y (7).
E755A, the negative charge reducing mut ation in domain II, increased lidocaine af fin it y, but D400A, a similar charge reducing mut ation of domain I, and A1529D, the negative residue replacement of domain I V, had no ef fect on af fin it y.
Consistent w ith this, D400A and A1529D create an ac cess pathway but do not af fect dr ug binding.
K1237R
protein
substitution
P15390
true positive
A n intermediate ef fect on the lidocaine block resulted f r om K1237S and there was no ef fect f r om K1237R, implying an elect r ost atic ef fect of Lys.
Substitution of Lys w ith a neutral residue, Ser (K1237S), shif ted the cur ve to an inter mediate position ( Kd 281 47 M) and substitution of Lys w ith the positively charged Arg (K1237R) resulted in w ild-t ype af fin it y (Kd 5 21 50 M; P 0.05 by Tukey test follow ing t wo-way ANOVA) (Fig.
Peak currents 10 11 min af ter ex posure to indicated c oncentrations of lidocaine were nor malized to that in the c ontrol for 1 w ild-t ype (W T, E), D400A (domain I, F), E755A (domain II, OE), K1237E (domain III, ) and A1529D (domain I V, ) in A and for W T (E), K1237E ( ), K1237S (OE) and K1237R (F) in B.
Kd values are 572, 552, 284, 154, 281, 521, and 611 M for W T, D400A, E755A, K1237E, K1237S, K1237R, and A1529D, respectively.
Nevertheless, K1237R increases the per meation of K (7), but it had no ef fect on lidocaine block, r uling out any role of K per meation.
A further support for an electrost atic ef fect of Lys at position 1,237 c omes f rom the absence of ef fects on lidocaine binding af fin it y w ith K1237R, where the negative charge at position 1,237 was preser ved.
K1237S
protein
substitution
P15390
true positive
A n intermediate ef fect on the lidocaine block resulted f r om K1237S and there was no ef fect f r om K1237R, implying an elect r ost atic ef fect of Lys.
Substitution of Lys w ith a neutral residue, Ser (K1237S), shif ted the cur ve to an inter mediate position ( Kd 281 47 M) and substitution of Lys w ith the positively charged Arg (K1237R) resulted in w ild-t ype af fin it y (Kd 5 21 50 M; P 0.05 by Tukey test follow ing t wo-way ANOVA) (Fig.
Interestingly, even though K1237 interacted electrost atically w ith lidocaine, neither K1237E nor K1237S allowed external QX222 block (Fig.
2D; 6.6 1.2% block, n 5 for K1237E, and 4.3 1.4% block, n 4 for K1237S).
Peak currents 10 11 min af ter ex posure to indicated c oncentrations of lidocaine were nor malized to that in the c ontrol for 1 w ild-t ype (W T, E), D400A (domain I, F), E755A (domain II, OE), K1237E (domain III, ) and A1529D (domain I V, ) in A and for W T (E), K1237E ( ), K1237S (OE) and K1237R (F) in B.
Kd values are 572, 552, 284, 154, 281, 521, and 611 M for W T, D400A, E755A, K1237E, K1237S, K1237R, and A1529D, respectively.
( D ) K1237E (E) and K1237S ( ).
The mut ation K1237S changed lidocaine af fin it y but did not af fect the volt age dependence of gating, however.
K1237E is per meable to Ba 2 (6), but K1237S seems to increase Ba2 block of Na currents because Na currents were dramatically reduced af ter changing to Ba 2 solution (dat a not shown).
If this af fin it y change was solely caused by electrost atic ef fect, the above equation predicts the Kd value of 297 M in K1237S, which was c onsistent w ith the obser ved Kd value of 281 M.
F1579A
protein
substitution
P15390
true positive
In c ontrast, the double mut ant E755A plus Y1586A showed sign ificantly reduced external block (10.1 1.1%, n 6) and E755A plus F1579A showed no detect able block (1.0 0.5%, n 5) during 11- to 12-min ex posure to external 500 M QX222 (P 0.001).
These c onclusions are derived f rom the f o l l o w i n g f i n d i n g s i n s e l e c t i v i t y f i l t e r m u t a n t s : (i ) a c c e l e r a t e d rec over y f rom internal QX block; (ii) a similar time c ourse of rec over y f rom QX block when QX was applied f rom the inside or the outside; (iii) no block or reduced block by external QX in double mut ant (E755A F1579A, E755A Y1586A); and (iv) outside QX ac cess impeded by the presence of outer vestibule 3.
A1529D
protein
substitution
P15390
true positive
Mut ation of the other selectivit y residues, D400A (domain I), E755A (domain II), and A1529D (domain I V) allowed block by exter nally applied quater nar y memb r a n e - i m p e r m e a n t d e r i v a t i v es o f l i d o c a i n e ( Q X 3 1 4 a n d QX222) and ac celerated rec over y f r om block by inter nal QX314.
Neo-sax itox in and tet r odotox in, w hich oc clude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively.
In w ild t ype, the lidocaine dissociation c onst ant (Kd) was 572 38 M and no change was obser ved in domain I mut ant D400A (Kd 552 39 M) or domain I V mut ant A1529D (Kd 611 35 M) (Fig.
2 B), and A1529D (Fig.
On average, block was 20.5 2.5% (n 9), 38.2 2.5% (n 8), and 29.2 2.6% (n 5) for D400A, E755A, and A1529D, respectively, af ter 11 min of per fusion.
E755A and A1529D also showed 32.1 2.5% (n 10) and 60.8 3.0% (n 5) block 10 min af ter external application of 500 M QX314.
Peak currents 10 11 min af ter ex posure to indicated c oncentrations of lidocaine were nor malized to that in the c ontrol for 1 w ild-t ype (W T, E), D400A (domain I, F), E755A (domain II, OE), K1237E (domain III, ) and A1529D (domain I V, ) in A and for W T (E), K1237E ( ), K1237S (OE) and K1237R (F) in B.
Kd values are 572, 552, 284, 154, 281, 521, and 611 M for W T, D400A, E755A, K1237E, K1237S, K1237R, and A1529D, respectively.
( C ) A1529D.
Peak currents elicited by 35-ms pulses to 10 mV f rom a holding potential of 100, 110 (for A1529D), or 120 mV (for K1237E) at 20-s inter vals were c or malized to peak current in c ontrol and plotted as relative Na n urrents (relative INa).
In c ontrast, the selectiv it y filter mut ants that per mitted block by external QX222, E755A, and A1529D showed much faster rec over y than that of w ild t ype (time c onst ants were 1,263 and 228 s, respectively).
A1529D also showed similar rec over y time c onst ants for internal (228 s) or external application (216 s) of QX314.
Twent y to 30 min af ter microinjection, a 1-Hz train of 20 pulses w ith a 35-ms duration was applied to 10 mV f rom a holding potential of 100 or 110 mV (for A1529D) to produce use-dependent block by QX314.
Degrees of use-dependent block by internal QX314 were 87.5, 78.3, 65.6, and 70.9% for w ild t ype, D400A, E755A, and A1529D, respectively.
The smooth lines are single ex ponential fits, and time c onst ants are 1,264 s for E755A and 228 s for A1529D.
For internal application, 50 nl of 3 mM QX222 or QX314 was microinjected into ooc y tes ex pressing E755A (E) and A1529D (,), respectively.
For external application, 500 M QX222 or 500 M QX314 was added to the bath solution for E755A (F) and A1529D (OE), respectively and per fused until rec over y protoc ol was c omplete.
The smooth lines are single ex ponential fits for E755A (solid lines) and A1529D (dashed lines) dat a.
For A1529D, time c onst ants are 228 s for internal and 216 s for external QX314.
In A1529D, 5 M T TX and the mixture of 5 M T TX and 500 M QX314 produced 100% block during a 5-min per fusion.
USA 94 (1997) 14129 upon washout of the mixture than that of QX314 alone was c onfir med in three and six additional ooc y tes for D400A and A1529D, respectively.
The graphs show t ypical examples of ex periments in D400A ( A ) and A1529D ( B ) .
The bar shows the period during ex posure to 1 M neo-STX (n-STX), the mixture of 1 M n-STX plus 1 mM QX314 (n-STX QX314), or 1 mM QX314 in D400A ( A ) ; and 500 M QX314, the mixture of 5 M T TX plus 500 M QX314 (T TX QX314), or 5 M T TX in A1529D ( B ) .
E755A increases K per meabilit y over Na (7) and A1529D is predicted to increase K per meabilit y f rom A1529E dat a (6).
Because K (radius 1.33 ) is bigger than 0.95 ), the selectiv it y filter region in E755A Na (radius and A1529D may have a larger diameter, and c onsequently, QX c ompounds might directly pass through it.
E755A, the negative charge reducing mut ation in domain II, increased lidocaine af fin it y, but D400A, a similar charge reducing mut ation of domain I, and A1529D, the negative residue replacement of domain I V, had no ef fect on af fin it y.
Consistent w ith this, D400A and A1529D create an ac cess pathway but do not af fect dr ug binding.
A1529E
protein
substitution
P15390
true positive
E755A increases K per meabilit y over Na (7) and A1529D is predicted to increase K per meabilit y f rom A1529E dat a (6).
10804199
full text
P1A
protein
substitution
true negative
T he GK domain of the PSD-95 family of M AGUK s interacts w ith the GK A P/SA PA P/ DA P family of postsy naptic densit y proteins (K im et al., 1997; Nai sbitt et al., 1997; Takeuchi et al., 1997), BEGA I N (Deguchi et al., 1998), and M A P1A (Brenman et al., 1998).
P542A
protein
substitution
true negative
pGA D10 constructs: SH3 domai ns: PSD-95 (aa 431500), SA P97 (aa 581 648 of hdlg), chapsy n-110 (aa 539 608 of human chapsy n-110); GK r: PSD-95 (aa 508 724), SA P97 (aa 656 911), dlg (aa 728 960); GK r poi nt mutations, PSD-95 P542A (proli ne-542 to alani ne), PSD-95 P569A , PSD-95 P719A , PSD-95 P542A P569A, PSD-95 P542A P569A P719A; SH3 GK (S G) domai ns: PSD-95 (aa 431724), SA P97 (aa 580 911), chapsy n-110 (aa 522 852).
SH3 binding was retained even w ith the double mutation (P542A / P569A) and the triple mutation (P542A / P569A / P719A) in the GK region, although the strength of interaction in the yeast t wo-hybrid system became weaker w ith increasing number of substitutions (Fig.
W470A
protein
substitution
true negative
GW1PSD-95s w ith poi nt mutations (L460P and W470A) were made usi ng QuickC hange site-di rected mutagenesi s k it (Stratagene).
pBH A constructs: SH3 domai ns: SA P97 (aa 580 652), chapsy n-110 (aa 539 608), dlg (aa 600 680), Z O-1 (aa 583 801); GK regions: PSD-95 (aa 508 724), SA P97 (aa 656 911), chapsy n-110 (aa 630 852); SH3 GK (S G) domains: PSD-95 (aa 431724), SA P97 (aa 580 911), chapsy n-110 (aa 522 852); SH3 GK mi ssing the last 13 amino acids (S G C): PSD-95 (aa 431711), SA P97 (aa 580 898); S G poi nt mutations: PSD-95 S G L460P, PSD-95 S G W470A .
Substitution mutants (L460P and W470A) of the SH3 domain of PSD-95 do not interact w ith GK regions from PSD95, SA P97, and chapsy n-110.
T he S G construct of PSD-95, w ith a L460P mutation in the SH3 domain (S* G(L460P)) can interact w ith the SH3 domain of PSD-95, whereas S* G(W470A) does not interact w ith the SH3 domains.
Mutating amino acid 470 in the SH3 domain of PSD-95 from tr y ptophan to alanine (W470A), a substitution k now n to prevent the interaction bet ween SH3 domains and proline-rich motifs, di srupted PSD-95-K A2 binding (Garcia et al., 1998).
We introduced the W470A mutation into the SH3 domain of PSD-95 and found that it aboli shed SH3 interaction w ith the GK regions of PSD-95 family proteins in yeast t wo-hybrid assays (Fig.
We have show n above that the W470A mutation in the SH3 domain of PSD-95 di srupts the intermolecular interaction bet ween SH3 domain and GK regions in the yeast t wo-hybrid assay (Fig.
Interestingly, a S G construct of PSD-95 containing the W470A mutation (S* G(W470A)) showed no interaction w ith SH3 domains from PSD-95 or chapsy n-110 while interacting robustly w ith GK A P (Table 1, row 5).
T hi s finding suggests that the W470A mutation did not di srupt the intramolecular interaction bet ween SH3 and GK r in the S G construct.
Finally, we tested the clustering behav ior of PSD-95 containing the W470A mutation.
It i s notable that PSD-95 w ith the W470A mutation (which has an intact intramolecular SH3 GK r interaction) showed association w ith N-PSD-95-GFP comparable to w ild-t y pe PSD-95 (Fig.
PSD-95(W470A) mutant, however, formed w ild-t y pe-looking clusters w ith Kv1.4 (bottom panels).
Nevertheless, the inhibitor y effect of the W470A mutation (equi valent to W118 of Src SH3 domain) on intermolecular SH3 binding to the GK region i s consi stent w ith the idea that the PSD-95 SH3 domain uses the equi valent peptide binding sur face as used by the Src SH3 domain.
Gi ven that interpretation, it i s significant that the same SH3 mutation (W470A) that aboli shes intermolecular SH3 GK r association does not prevent intramolecular SH3 GK r interaction.
P569A
protein
substitution
true negative
pGA D10 constructs: SH3 domai ns: PSD-95 (aa 431500), SA P97 (aa 581 648 of hdlg), chapsy n-110 (aa 539 608 of human chapsy n-110); GK r: PSD-95 (aa 508 724), SA P97 (aa 656 911), dlg (aa 728 960); GK r poi nt mutations, PSD-95 P542A (proli ne-542 to alani ne), PSD-95 P569A , PSD-95 P719A , PSD-95 P542A P569A, PSD-95 P542A P569A P719A; SH3 GK (S G) domai ns: PSD-95 (aa 431724), SA P97 (aa 580 911), chapsy n-110 (aa 522 852).
SH3 binding was retained even w ith the double mutation (P542A / P569A) and the triple mutation (P542A / P569A / P719A) in the GK region, although the strength of interaction in the yeast t wo-hybrid system became weaker w ith increasing number of substitutions (Fig.
L460P
protein
substitution
true negative
GW1PSD-95s w ith poi nt mutations (L460P and W470A) were made usi ng QuickC hange site-di rected mutagenesi s k it (Stratagene).
Br iefly, C OS c el ls si ng l y transfected w ith PSD-95 W T, L460P, or C were ex tracted w ith 1% Tr iton X-100, fol lowed by i ncubation w ith GST-f usion protei ns and precipitation w ith g lutathione Sepharose 4B resi n (A mersham Pharmacia Biotech).
pBH A constructs: SH3 domai ns: SA P97 (aa 580 652), chapsy n-110 (aa 539 608), dlg (aa 600 680), Z O-1 (aa 583 801); GK regions: PSD-95 (aa 508 724), SA P97 (aa 656 911), chapsy n-110 (aa 630 852); SH3 GK (S G) domains: PSD-95 (aa 431724), SA P97 (aa 580 911), chapsy n-110 (aa 522 852); SH3 GK mi ssing the last 13 amino acids (S G C): PSD-95 (aa 431711), SA P97 (aa 580 898); S G poi nt mutations: PSD-95 S G L460P, PSD-95 S G W470A .
Substitution mutants (L460P and W470A) of the SH3 domain of PSD-95 do not interact w ith GK regions from PSD95, SA P97, and chapsy n-110.
T he S G construct of PSD-95, w ith a L460P mutation in the SH3 domain (S* G(L460P)) can interact w ith the SH3 domain of PSD-95, whereas S* G(W470A) does not interact w ith the SH3 domains.
A n equi valent point mutation (leucine to proline) in residue 460 of PSD-95 (L460P) di srupted the interaction bet ween the SH3 domain of PSD-95 and the GK region of PSD-95 family proteins (Fig.
Similarly, an S G construct of PSD-95 w ith a point mutation (L460P) in the SH3 domain (S* G(L460P)) interacted in trans w ith the SH3 domain of PSD-95, but not w ith the GK region (Table 1, row 4).
T hi s i s the ex pected result if the L460P mutation di srupted the intramolecular interaction bet ween SH3 domain and GK region, liberating the intact GK region for binding in trans to a w ild-t y pe SH3 domain.
T he binding bet ween S* G(L460P) and PSD-95 SH3 domain appeared weaker than bet ween the i solated GK region and SH3 domain (Table 1, Fig.
We could not detect an interaction bet ween PSD-95 S* G(L460P) and the SH3 domain of chapsy n-110, perhaps related to the fact that the interaction bet ween the GK r of PSD-95 and the SH3 domain of chapsy n-110 was already weaker than w ith the PSD-95 SH3 domain (Fig.
When ex pressed in C OS cells, the SH3 domain mutant PSD-95(L460P), but not w ild-t y pe PSD-95 or PSD-95 C, could be "pulled dow n" by GST-f usion protein of the SH3 domain of PSD-95 (Fig.
T hese results are ex plained if the L460P mutation di srupts intramolecular SH3 GK r interaction, thereby freeing the intact GK region to bind to GST-SH3.
Similarly, PSD-95 C, but not w ild-t y pe PSD-95 or PSD95(L460P), was pulled dow n by GST-f usion protein containing the GK region of PSD-95 (Fig.
Further supporting an intramolecular SH3 GK r interaction, GST-f usion protein of the combined SH3 and GK regions (S G) could not pull-dow n any PSD-95 protein ex pressed in C OS cells, whether w ild-t y pe or L460P (Fig.
In C OS cells doubly transfected w ith K channel Kv1.4 and PSD-95 or PSD-95(L460P) or PSD95 C, the amount of Kv1.4 coimmunoprecipitated w ith PSD-95 antibodies was not significantly different bet ween w ild-t y pe and mutant PSD-95 proteins (Fig.
T hus, di srupting the intramolecular SH3 GK r interaction by mutating the SH3 domain (L460P) or the GK region ( C) had no obv ious effect on the association of PSD-95 w ith its binding partners Kv1.4 or GK A P in mammalian cells.
Interestingly, C OS cells doubly transfected w ith Kv1.4 and PSD-95(L460P) or w ith Kv1.4 and PSD-95 C did not form any t y pical clusters.
Because both the L460P and C mutations impai r SH3 GK r interaction, but not Kv1.4 binding by PSD-95, these results suggest that the S G intramolecular interaction i s important for the ion channel-clustering acti v it y of PSD-95.
To probe the mechani sm underly ing the loss of channelclustering acti v it y of PSD-95 mutants L460P and C, we tested these mutant proteins for multimeri z ation and membrane association, which are important steps in the process of Kv1.4 clustering by PSD-95 (H sueh et al., 1997; Topinka and Bredt, 1998; H sueh and Sheng, 1999).
In a C OS cell coimmunoprecipitation assay, PSD-95 mutants L460P and C were able to associate w ith a PSD-95-GFP-f usion protein containing the N-terminal multimeri z ation domain of PSD-95 (N-PSD-95-GFP; Fig.
T he coimmunoprecipitation of these PSD-95 mutants (L460P and C) w ith N-PSD-95-GFP was actually better than w ild-t y pe PSD-95 (Fig.
Wild-t y pe and mutant (L460P and C) PSD-95 proteins ex pressed in C OS cells were incubated w ith GST-f usion proteins of the PSD-95 SH3 domain, GK region, or combined SH3 GK (SG) regions, or w ith GST alone.
C OS cells doubly transfected w ith Kv1.4 and w ild-t y pe or mutant (L460P and C) PSD-95 were immunoprecipitated w ith PSD-95 antibody, and the precipitates immunoblotted for Kv1.4 and PSD-95.
T y pical plaque-like coclusters formed bet ween Kv1.4 and w ild-t y pe PSD-95 (top panels), but were not seen w ith PSD-95 mutants L460P and C (middle panels).
In membrane fractionation ex periments, L460P and C mutants of PSD-95 ex pressed in C OS cells showed membrane association and membrane ex tractabilit y properties that were similar to w ild-t y pe PSD-95 (Fig.
Taken together, these results indicate that the loss of clustering acti v it y in PSD-95 L460P and C cannot be attributed simply to loss of multimeri z ation or membrane association of these PSD-95 mutants.
B, C OS cells were transfected w ith w ildt y pe or mutant (L460P or C) PSD-95.
T hi s effect i s relati vely specific because the same mutations in the PSD-95 protein (L460P and C) had no effect on Kv1.4 binding or membrane association of PSD-95; nor did they di srupt head-tohead multimeri z ation of PSD-95.
Because, if any thing, the L460P and C mutations would enhance cross-linking of PSD-95 by promoting intermolecular (trans) SH3 GK association, thei r deleterious effects specifically on channel clustering carries more significance.
Our observation that L460P and C mutants often form perinuclear clusters w ith Kv1.4 rai ses the additional possibilit y that an intact SH3 GK r interaction i s requi red for proper intracellular tra fficking of PSD-95 (A rnold and C lapham, 1999; Craven et al., 1999; ElHusseini et al., 2000; Tiffany et al., 2000).
W470F
protein
substitution
true negative
T hese investigators found that a W470F mutation did not aboli sh intermolecular SH3 GK interaction.
D95C
protein
substitution
true negative
In C OS cells doubly transfected w ith K channel Kv1.4 and PSD-95 or PSD-95(L460P) or PSD95 C, the amount of Kv1.4 coimmunoprecipitated w ith PSD-95 antibodies was not significantly different bet ween w ild-t y pe and mutant PSD-95 proteins (Fig.
P719A
protein
substitution
true negative
pGA D10 constructs: SH3 domai ns: PSD-95 (aa 431500), SA P97 (aa 581 648 of hdlg), chapsy n-110 (aa 539 608 of human chapsy n-110); GK r: PSD-95 (aa 508 724), SA P97 (aa 656 911), dlg (aa 728 960); GK r poi nt mutations, PSD-95 P542A (proli ne-542 to alani ne), PSD-95 P569A , PSD-95 P719A , PSD-95 P542A P569A, PSD-95 P542A P569A P719A; SH3 GK (S G) domai ns: PSD-95 (aa 431724), SA P97 (aa 580 911), chapsy n-110 (aa 522 852).
SH3 binding was retained even w ith the double mutation (P542A / P569A) and the triple mutation (P542A / P569A / P719A) in the GK region, although the strength of interaction in the yeast t wo-hybrid system became weaker w ith increasing number of substitutions (Fig.
11553787
full text
R318Q
protein
substitution
true positive
O88703
Mutation of a basic residue in the S4 domain (R318Q) prevented channel opening, presumably by disrupting S4 movement.
However, channels with R318Q and Y331S mutations were constitutively open, suggesting that these channels can open without a functioning S4 domain.
R318Q Y331S ntHCN2 Channels Are Constitutively Open.
In a prev ious study, we found that channels hav ing a mut ation of a basic residue (R318Q) in the S4 domain of ntHCN2 failed to open in response to membrane depolarization, yet mut ant protein was still ex pressed at the sur face membrane about half as ef fectively as W T HCN2 protein (19).
If Y331 mut ations disr upt channel closure by 11280 www.pnas.org cgi doi 10.1073 pnas.201250598 inter fering w ith the nor mal c oupling bet ween volt agedependent S4 movement and closure of the activation gate, then we predicted that the sec ond mut ation Y331S c ould rescue the function of R318Q ntHCN2 channels.
Addition of a sec ond mut ation (R318Q Y331S) resulted in functional ex pression.
A lthough R318Q Y331S ntHCN2 channels appeared to be c onstitutively open, the IV relationship still exhibited rectification (Fig.
R318Q Y331S ntHCN2 channels are constitutively open.
(A) R318Q ntHCN2 channel currents were undetectable.
(B) R318Q Y331S ntHCN2 channel currents activate instantaneously and have no time-dependent component, unlike Y331S channel currents (Fig.
(C) Currentvoltage relationships for R318Q and R318Q Y331S HCN2 channel currents.
We tested this hypothesis by c omparing the function of channels c ont ain ing a single S4 domain mut ation (R318Q) or a double mut ation (R318Q Y331S).
In our prev ious study of the Hackos, S4 domain of HCN2 channels (19), we found that the R318Q mut ation abolished function, but did not prevent traf fick ing and insertion of the channels into the plasma membrane, suggesting that loss of function was caused by a failure of the S4 domain to move properly in response to a change in transmembrane potential.
Introduction of a sec ond mut ation (Y331S) into R318Q HCN2 rescued function, but also changed the gating in an import ant way.
R318Q Y331S channels were always open, 1 av ing no time-dependent c omponent of current (i.e., min-Po h ).
The lack of time-dependent gating of R318Q Y331S channels suggests a disc onnection bet ween the nor mal requirement for volt age sensing and channel activation; however, we cannot r ule out the possibilit y that addition of the Y331S mut ation to R318Q HCN2 locks the S4 domain in the activated position.
D540K
protein
substitution
true positive
Q12809
In addition, one mut ation (D540K) disr upted channel closure and caused the channel to open slowly in response to hyperpolarization, similar to activation of HCN channel current.
Hyperpolarization-dependent gating of D540K HERG channels was steeply volt age-dependent and saturated at 160 mV (24), suggesting that inward movement of the S4 domain mediated the hyperpolarization-induced channel open ing.
We hypothesized that, similar to D540K HERG, the S4 S5 linker might be a cr ucial link in the hyperpolarization-dependent gating pathway of HCN channels.
On the basis of our obser vation that HERG channels c ont ain ing a single mut ation of the S4 S5 linker (D540K) disr upt the closed st ate and allow reopen ing in response to hyperpolarization (24), we hypothesized that mut ations in the S4 S5 linker of HCN2 channels might also alter channel gating.
D332A
protein
substitution
true positive
O88703
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
In addition, mut ation of single amino acids near this Ty r residue had no sign ificant af fect on gating (e.g., T330A, D332A).
S335A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
R339C
protein
substitution
true positive
O88703
As ex pected, R339Q and R339C ntHCN2 channels c ould not close properly (Fig.
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
(A) Representative current traces for R339A, R339Q, R339C, R339E, and R339D ntHCN2 channels.
Holding potential was 0 mV and test potentials ranged from 120 to 40 mV for R339Q, R339C, and R339E ntHCN2 channels.
R339A
protein
substitution
true positive
O88703
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
The min-Po was 0.31 0.003 for E324A (n 11), 0.58 0.005 for Y331A (n 9), and 0.34 0.003 for R339A (n 11) ntHCN2 channels.
(A) Representative current traces for R339A, R339Q, R339C, R339E, and R339D ntHCN2 channels.
Holding potential was 30 mV and test potentials ranged from 140 to 30 mV for R339A and R339D.
R339D
protein
substitution
true positive
O88703
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
(A) Representative current traces for R339A, R339Q, R339C, R339E, and R339D ntHCN2 channels.
Holding potential was 30 mV and test potentials ranged from 140 to 30 mV for R339A and R339D.
In c ontrast, the min-Po for R339D was 0.17 0.004 (n 8), only about t w ice as large as W T ntHCN2 channels.
H328A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
Q322A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
E324D
protein
substitution
O88703
true positive
The most c onser vative mut ation, E324D, resulted in a nonfunctional channel as judged by lack of measurable currents.
R339Q
protein
substitution
true positive
O88703
As ex pected, R339Q and R339C ntHCN2 channels c ould not close properly (Fig.
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
(A) Representative current traces for R339A, R339Q, R339C, R339E, and R339D ntHCN2 channels.
Holding potential was 0 mV and test potentials ranged from 120 to 40 mV for R339Q, R339C, and R339E ntHCN2 channels.
E324A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
The min-Po was 0.31 0.003 for E324A (n 11), 0.58 0.005 for Y331A (n 9), and 0.34 0.003 for R339A (n 11) ntHCN2 channels.
(A) Representative current traces for E324A, E324Q, and E324K ntHCN2 channels.
The holding potential was 0 mV and test potentials were from 140 to 0 mV for E324Q and 120 to 0 mV for E324A and E324K.
Because the min-Po of E324A ntHCN2 channels was increased and had a V1/2 for activation shif ted by 30 mV, we ex plored the ef fects of other mut ations of this residue (Fig.
I340A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
E324K
protein
substitution
O88703
true positive
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
(A) Representative current traces for E324A, E324Q, and E324K ntHCN2 channels.
The holding potential was 0 mV and test potentials were from 140 to 0 mV for E324Q and 120 to 0 mV for E324A and E324K.
M329A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
V337A
protein
substitution
true positive
O88703
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
F327A
protein
substitution
O88703
true positive
(B) Representative whole-cell recordings of WT, F327A, and W323A ntHCN2 channel currents elicited with 3-s pulses, applied from a holding potential of 30 mV in 10-mV increments to potentials ranging from 140 to 30 mV.
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
In most cases, mut ation of a single S4 S5 linker amino acid to A la af fected the volt age dependence or k inetics of ntHCN2 current activation (e.g., F327A, W323A, Fig.
W323A
protein
substitution
O88703
true positive
(B) Representative whole-cell recordings of WT, F327A, and W323A ntHCN2 channel currents elicited with 3-s pulses, applied from a holding potential of 30 mV in 10-mV increments to potentials ranging from 140 to 30 mV.
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
In most cases, mut ation of a single S4 S5 linker amino acid to A la af fected the volt age dependence or k inetics of ntHCN2 current activation (e.g., F327A, W323A, Fig.
Y331D
protein
substitution
true positive
O88703
(A) Representative current traces for Y331A, Y331S, Y331D, and Y331F ntHCN2 channels.
Holding potential was 0 mV and test potentials were 120 to 40 mV for Y331A, Y331S, and Y331D channels.
The inst ant aneous currents c onducted by Y331S, Y331D (Fig.
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
The large inst ant aneous and small time-dependent c omponent of current for Y331S, Y331A, Y331D, and Y331K ntHCN2 channels suggested that channels remained largely open at 0 mV.
P64T
protein
substitution
true positive
Q64018
Materials and Methods HCN2 channel cDNA was cloned f rom Marathon-Ready (CLONTECH) mouse brain cDNA into the pSP64T ooc y te ex pression vector (19).
Y331F
protein
substitution
true positive
O88703
(A) Representative current traces for Y331A, Y331S, Y331D, and Y331F ntHCN2 channels.
Holding potential was 30 mV and test potentials were 140 to 30 mV for Y331F channels.
In c ontrast, Y331F channels opened and closed relatively nor mally (Fig.
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
Y331A
protein
substitution
true positive
O88703
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
(A) Representative current traces for Y331A, Y331S, Y331D, and Y331F ntHCN2 channels.
Holding potential was 0 mV and test potentials were 120 to 40 mV for Y331A, Y331S, and Y331D channels.
The min-Po was 0.31 0.003 for E324A (n 11), 0.58 0.005 for Y331A (n 9), and 0.34 0.003 for R339A (n 11) ntHCN2 channels.
Y331A ntHCN2 channel currents were rec orded at test summar y, an aromatic residue at position 331 in the S4 S5 linker of HCN2 channels was required for nor mal channel gating.
Unlike W T channels, Y331A ntHCN2 channels c onducted sign ificant outward currents at potentials positive to the reversal potential of 30 mV.
T h e V 1/2 f o r v o l t a g e - d e p e n d e n t activation of Y331A and Y331S channels was shif ted to more positive potentials and the slope factor increased c ompared w ith W T ntHCN2 channels (Tables 1 and 2), but these changes c ould not ac c ount for the dramatic increase in min-Po (Fig.
The large inst ant aneous and small time-dependent c omponent of current for Y331S, Y331A, Y331D, and Y331K ntHCN2 channels suggested that channels remained largely open at 0 mV.
E324Q
protein
substitution
O88703
true positive
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
(A) Representative current traces for E324A, E324Q, and E324K ntHCN2 channels.
The holding potential was 0 mV and test potentials were from 140 to 0 mV for E324Q and 120 to 0 mV for E324A and E324K.
R339E
protein
substitution
true positive
O88703
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
(A) Representative current traces for R339A, R339Q, R339C, R339E, and R339D ntHCN2 channels.
Holding potential was 0 mV and test potentials ranged from 120 to 40 mV for R339Q, R339C, and R339E ntHCN2 channels.
The min-Po for R339E was 0.65 0.002 (n 12).
Y331K
protein
substitution
true positive
O88703
2 A), and Y331K (not shown) ntHCN2 channels were even g reater than o b s e r v e d f o r Y 3 3 1 A c h a n n e l s .
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
The large inst ant aneous and small time-dependent c omponent of current for Y331S, Y331A, Y331D, and Y331K ntHCN2 channels suggested that channels remained largely open at 0 mV.
E325A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
Y331S
protein
substitution
true positive
O88703
However, channels with R318Q and Y331S mutations were constitutively open, suggesting that these channels can open without a functioning S4 domain.
(A) Representative current traces for Y331A, Y331S, Y331D, and Y331F ntHCN2 channels.
Holding potential was 0 mV and test potentials were 120 to 40 mV for Y331A, Y331S, and Y331D channels.
The inst ant aneous currents c onducted by Y331S, Y331D (Fig.
T h e V 1/2 f o r v o l t a g e - d e p e n d e n t activation of Y331A and Y331S channels was shif ted to more positive potentials and the slope factor increased c ompared w ith W T ntHCN2 channels (Tables 1 and 2), but these changes c ould not ac c ount for the dramatic increase in min-Po (Fig.
While this was tr ue for R339E, it was not the case for R339D Mutant Y331S Y331D Y331K Y331F R339E R339Q R339C R339D E324Q E324K V1/2, mV 76.0 80.7 75.4 99.8 56.6 64.4 62.4 74.8 75.4 78.7 1.0 4.0 1.3 0.8 0.9 1.1 1.5 0.5 1.3 0.6 k, mV 10.5 7.7 7.7 5.7 12.8 12.8 12.4 7.5 7.7 7.4 1.0 4.1 0.8 0.1 0.8 0.8 0.6 0.4 0.4 0.2 n 6 22 11 7 12 22 13 8 6 13 PNAS September 25, 2001 vol.
R318Q Y331S ntHCN2 Channels Are Constitutively Open.
The large inst ant aneous and small time-dependent c omponent of current for Y331S, Y331A, Y331D, and Y331K ntHCN2 channels suggested that channels remained largely open at 0 mV.
If Y331 mut ations disr upt channel closure by 11280 www.pnas.org cgi doi 10.1073 pnas.201250598 inter fering w ith the nor mal c oupling bet ween volt agedependent S4 movement and closure of the activation gate, then we predicted that the sec ond mut ation Y331S c ould rescue the function of R318Q ntHCN2 channels.
Addition of a sec ond mut ation (R318Q Y331S) resulted in functional ex pression.
However, unlike Y331S ntHCN2 channel current, steps in membrane potential elicited inst ant aneous currents w ithout a time-independent c omponent (Fig.
A lthough R318Q Y331S ntHCN2 channels appeared to be c onstitutively open, the IV relationship still exhibited rectification (Fig.
R318Q Y331S ntHCN2 channels are constitutively open.
(B) R318Q Y331S ntHCN2 channel currents activate instantaneously and have no time-dependent component, unlike Y331S channel currents (Fig.
(C) Currentvoltage relationships for R318Q and R318Q Y331S HCN2 channel currents.
We tested this hypothesis by c omparing the function of channels c ont ain ing a single S4 domain mut ation (R318Q) or a double mut ation (R318Q Y331S).
Introduction of a sec ond mut ation (Y331S) into R318Q HCN2 rescued function, but also changed the gating in an import ant way.
R318Q Y331S channels were always open, 1 av ing no time-dependent c omponent of current (i.e., min-Po h ).
By c ontrast, Y331S ntHCN2 channel current exhibited a time-dependent c omponent of current, albeit reduced c ompared w ith w ild-t ype current.
The lack of time-dependent gating of R318Q Y331S channels suggests a disc onnection bet ween the nor mal requirement for volt age sensing and channel activation; however, we cannot r ule out the possibilit y that addition of the Y331S mut ation to R318Q HCN2 locks the S4 domain in the activated position.
L333A
protein
substitution
true positive
O88703
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
T330A
protein
substitution
true positive
O88703
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
In addition, mut ation of single amino acids near this Ty r residue had no sign ificant af fect on gating (e.g., T330A, D332A).
M338A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
I326A
protein
substitution
O88703
true positive
Voltage dependence and kinetics of current activation for HCN2 channels containing Ala point mutations in the S4 S5 linker Channel ntHCN2 Q322A W323A E324A E325A I326A F327A H328A M329A T330A Y331A D332A L333A S335A V337A M338A R339A I340A V1/2, mV 84.2 81.8 68.1 55.0 82.2 80.8 98.8 109 80.1 83.7 62.9 72.3 73.8 81.6 90.0 87.8 72.5 96.6 0.2 0.7 1.2 0.9 1.0 0.5 0.8 0.3 1.2 0.2 1.2 0.6 0.9 0.4 0.8 1.1 1.3 1.1 k, mV 7.7 8.3 6.4 8.4 7.7 8.5 8.2 7.1 8.4 7.5 13.4 7.0 7.1 7.5 8.0 8.9 8.6 7.3 0.1 0.1 0.3 0.4 0.3 0.3 0.3 0.7 0.5 0.2 1.0 0.2 0.8 0.3 0.4 0.3 0.4 0.5 n 8 9 8 10 8 10 7 5 4 10 9 12 8 5 4 6 11 5 act, ms 342 489 180 172 311 365 721 826 185 313 203 160 223 275 454 517 248 535 24 27 19 7 19 14 54 80 16 13 10 5 9 78 11 22 13 16 n 18 9 8 8 8 8 7 5 6 10 6 8 9 6 6 8 10 11 V1/2, potential of half-maximal current activation; k, slope factor of the activation curve; act, time constant for current activation at 120 mV; n, number of oocytes.
10354438
full text
Y1586A
protein
substitution
true positive
P15390
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
For comparison, 1-Y1586A mutant channels, like 1-Y1586K, are again BTX-sensitive (Fig.
These results suggest that the positive charge brought in by the lysine mutation at the 1-F1579 position may be crucial for the BTX-resistant phenotype, but less so at the 1-N1584 Wang and Wang BTX-Resistant Na channels 3145 FIGURE 4 Superimposed current traces were recorded from cells expressing 1-F1579A (A), 1-N1584A (B), 1-Y1586A (C), and hH1-N1765A (D).
The RBIIA-Y1771A mutant channel, like 1-Y1586A (Fig.
I1575D
protein
substitution
true positive
P15390
Mutants of 1-I1575K and I1575D were prepared, but they failed to express sufficient Na currents for the determination of the BTX phenotype.
I1575K
protein
substitution
true positive
P15390
Mutants of 1-I1575K and I1575D were prepared, but they failed to express sufficient Na currents for the determination of the BTX phenotype.
Y1586K
protein
substitution
true positive
P15390
In contrast, 1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization.
The third lysine point mutation located at the LA binding site, Y1586K, remains BTX sensitive.
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
This procedure was particularly important for cells transfected with 1-Y1586K, in which a significant number of channels were inactivated at 100 mV (Wright et al., 1998).
The third lysine mutant that affects the LA binding is 1-Y1586K, which expresses Na currents with normal kinetics (Fig.
Internal BTX at 5 M readily modifies the current of 1-Y1586K (Fig.
3144 Biophysical Journal Volume 76 June 1999 FIGURE 3 Families of Na currents from cells expressing 1-N1584K (A) and 1-Y1586K mutant (B) channels were superimposed.
Current traces of 1-N1584K (C) and 1-Y1586K (D) were superimposed along with the numbers corresponding to the number of pulses applied.
amplitude of 1-Y1586K BTX-modified Na current during repetitive pulses grows larger than the original peak current amplitude.
BTX also shifts the activation threshold of 1-Y1586K significantly, by 40 mV toward the hyperpolarizing direction.
Thus two out of three residues critical for LA binding ( 1-F1579K and 1-N1584K but not 1-Y1586K) display a complete BTXresistant phenotype after the lysine substitution.
For comparison, 1-Y1586A mutant channels, like 1-Y1586K, are again BTX-sensitive (Fig.
3146 Biophysical Journal Volume 76 June 1999 FIGURE 6 Time-dependent block of Na currents after 300 M cocaine application was recorded from cells expressing 1-wild-type (A), 1-F1579A (B), 1-N1584D (C), and 1Y1586K currents.
The numbers of experiments for wild-type, F1579A, N1584A, and Y1586K are 7, 6, 8, and 5, respectively.
State-dependent cocaine block of the BTXmodified mutants 1-F1579A, 1-N1584D, and 1-Y1586K As expected, the cocaine affinity for the open channel of BTX-modified 1-F1579A is diminished significantly (Fig.
Unexpectedly, we found that the state-dependent block of BTX-modified 1-Y1586K channels by 300 M cocaine was stronger than that of their wild-type counterparts (Fig.
Without BTX, the cocaine binding affinity in 1-Y1586K channels was recently reported to be identical to that in the wild-type channels in their resting state, whereas the benzocaine affinity was increased (Wright et al., 1998).
This cation- interaction may be facilitated by channel opening, because the cocaine-induced time-dependent block is significantly faster in 1-Y1586K mutant channels ( 71.2 12.2 ms, n 5) than in wild-type channels ( 177.5 18.2 ms, n 7, p 0.05).
F1579A
protein
substitution
P15390
true positive
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
As in brain Na channels for homologous mutations, the point mutation at 1-F1579A reduces LA binding, whereas the point mutation at 1-N1584A drastically enhances LA binding (Wang et al., 1998b).
The 1-F1579A mutant channels are readily modified by BTX, whereas the 1-N1584A channels remain relatively BTX resistant (Fig.
These results suggest that the positive charge brought in by the lysine mutation at the 1-F1579 position may be crucial for the BTX-resistant phenotype, but less so at the 1-N1584 Wang and Wang BTX-Resistant Na channels 3145 FIGURE 4 Superimposed current traces were recorded from cells expressing 1-F1579A (A), 1-N1584A (B), 1-Y1586A (C), and hH1-N1765A (D).
Peak current amplitudes in 1-F1579A, 1-N1584A, and hH1-N1765A were significantly reduced during repetitive pulses.
3146 Biophysical Journal Volume 76 June 1999 FIGURE 6 Time-dependent block of Na currents after 300 M cocaine application was recorded from cells expressing 1-wild-type (A), 1-F1579A (B), 1-N1584D (C), and 1Y1586K currents.
The numbers of experiments for wild-type, F1579A, N1584A, and Y1586K are 7, 6, 8, and 5, respectively.
This phenotype is very different from that of homologous 1-F1579A (Fig.
Why does 1-F1579A mutant, unlike RBIIA-F1764A, display a BTX-sensitive phenotype? Preliminary results showed that cardiac homologous mutant channel hH1F1760A is also BTX sensitive (n 5).
5 B) than that of 1-F1579A (Fig.
Our results thus suggest that these variable BTX phenotypes of homologous RBIIAF1764A, 1-F1579A, and hH1-F1760A mutants are likely isoform specific.
State-dependent cocaine block of the BTXmodified mutants 1-F1579A, 1-N1584D, and 1-Y1586K As expected, the cocaine affinity for the open channel of BTX-modified 1-F1579A is diminished significantly (Fig.
Such a reduction in LA affinity is found in resting and inactivated 1-F1579A Na channels in the absence of BTX (Wang et al., 1998b).
Because cocaine still elicits a time-dependent block in the open form of BTX-modified 1-F1579A mutant channels (Fig.
As in BTX-modified 1-F1579A, cocaine block is substantially reduced in BTX-modified 1-N1584D channels (Fig.
N1765A
protein
substitution
true positive
P15390
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
These results suggest that the positive charge brought in by the lysine mutation at the 1-F1579 position may be crucial for the BTX-resistant phenotype, but less so at the 1-N1584 Wang and Wang BTX-Resistant Na channels 3145 FIGURE 4 Superimposed current traces were recorded from cells expressing 1-F1579A (A), 1-N1584A (B), 1-Y1586A (C), and hH1-N1765A (D).
Peak current amplitudes in 1-F1579A, 1-N1584A, and hH1-N1765A were significantly reduced during repetitive pulses.
Expression of 1-N1584A and hH1-N1765A was generally poor in most cells ( 1 nA).
4 D shows that the hH1-N1765A current indeed displays a partially BTXresistant phenotype similar to that of the 1-N1584A cur- rent; a slower decaying phase is again evident after repetitive pulses.
Y1771A
protein
substitution
P04775
true positive
The RBIIA-Y1771A mutant channel, like 1-Y1586A (Fig.
N1584K
protein
substitution
P15390
true positive
We show that 1-F1579K and 1-N1584K channels become completely resistant to 5 M BTX.
In this report we show that the lysine point mutations of 1-F1579K and 1-N1584K at and near the LA binding site, respectively, can indeed affect the binding of BTX.
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
Another lysine mutant, 1-N1584K, also expresses Na urrents that are completely resistant to 5 M BTX.
3 A shows the current family of 1-N1584K at various voltages in the absence of BTX.
Nor did an increase in pulse number to 3000 alter this BTX-resistant phenotype in 1-N1584K.
3144 Biophysical Journal Volume 76 June 1999 FIGURE 3 Families of Na currents from cells expressing 1-N1584K (A) and 1-Y1586K mutant (B) channels were superimposed.
Notice that there are substantial late currents remaining at the end of depolarization in 1-N1584K mutant channels.
Current traces of 1-N1584K (C) and 1-Y1586K (D) were superimposed along with the numbers corresponding to the number of pulses applied.
Thus two out of three residues critical for LA binding ( 1-F1579K and 1-N1584K but not 1-Y1586K) display a complete BTXresistant phenotype after the lysine substitution.
By this argument we may tentatively equate the BTX binding affinity with the BTX-resistant phenotype in the following rank order: wild type 11-N1584K.
6) and that point mutations at 1-F1579K and 1-N1584K render channels resistant to BTX (Figs.
Wang and Wang BTX-Resistant Na channels 3149 and 1-N1584K) or a partial BTX-resistant phenotype ( 1N1584A and 1-N1584D).
F1764A
protein
substitution
P04775
true positive
It is noteworthy that RBIIA-F1764A of the rat brain Na hannel was found to exhibit a complete BTX-resistant phenotype under voltage-clamp conditions (Linford et al., 1998).
In fact, the current of this RBIIA-F1764A mutant is the only alanine mutant current that shows a complete resistance to 10 M BTX.
Why does 1-F1579A mutant, unlike RBIIA-F1764A, display a BTX-sensitive phenotype? Preliminary results showed that cardiac homologous mutant channel hH1F1760A is also BTX sensitive (n 5).
Our results thus suggest that these variable BTX phenotypes of homologous RBIIAF1764A, 1-F1579A, and hH1-F1760A mutants are likely isoform specific.
N1769A
protein
substitution
P04775
true positive
(1998) did not report the BTX phenotype of RBIIA-N1769A in their study.
N1584D
protein
substitution
P15390
true positive
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
We have found that 1-N1584D channels (substituted with the negatively charged residue aspartate) display an intermediate BTX-resistant phenotype (Fig.
The apparent fast decaying phase for 1-N1584D current has a FIGURE 5 Superimposed current traces of 1-wild-type (A) and 1-N1584D (B) were recorded in a slow time frame under reversed Na gradient conditions.
The threshold for channel activation is about 70 mV for 1-wild-type (A) and 1-N1584D mutant (B).
Notice that a large fraction of BTX-modified 1N1584D current was inactivated during prolonged depolarization.
3146 Biophysical Journal Volume 76 June 1999 FIGURE 6 Time-dependent block of Na currents after 300 M cocaine application was recorded from cells expressing 1-wild-type (A), 1-F1579A (B), 1-N1584D (C), and 1Y1586K currents.
Direct BTX bindN1584D 1-N1584A ing studies will be required to confirm this inference.
However, its phenotype is more similar to that of 1-N1584D (Fig.
State-dependent cocaine block of the BTXmodified mutants 1-F1579A, 1-N1584D, and 1-Y1586K As expected, the cocaine affinity for the open channel of BTX-modified 1-F1579A is diminished significantly (Fig.
As in BTX-modified 1-F1579A, cocaine block is substantially reduced in BTX-modified 1-N1584D channels (Fig.
Wang and Wang BTX-Resistant Na channels 3149 and 1-N1584K) or a partial BTX-resistant phenotype ( 1N1584A and 1-N1584D).
F1760A
protein
substitution
true positive
Q14524
Why does 1-F1579A mutant, unlike RBIIA-F1764A, display a BTX-sensitive phenotype? Preliminary results showed that cardiac homologous mutant channel hH1F1760A is also BTX sensitive (n 5).
Our results thus suggest that these variable BTX phenotypes of homologous RBIIAF1764A, 1-F1579A, and hH1-F1760A mutants are likely isoform specific.
Based on the complete BTX-resistant phenotype of homologous RBIIF1760A, this residue was suggested to interact directly with BTX (Linford et al., 1998).
F1579K
protein
substitution
P15390
true positive
We show that 1-F1579K and 1-N1584K channels become completely resistant to 5 M BTX.
In this report we show that the lysine point mutations of 1-F1579K and 1-N1584K at and near the LA binding site, respectively, can indeed affect the binding of BTX.
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
The mutant 1-F1579K channels exhibit current kinetics similar to those of 1 wild type (Fig.
In contrast, BTX failed to modify the 1-F1579K channels in all cells tested (n 10) after 1000 repetitive pulses under identical conditions.
In the presence of BTX, the FIGURE 2 Families of Na currents were recorded from cells expressing 1wild-type (A) and 1-F1579K mutant (B) channels under reversed Na gradient conditions.
In separate cells, BTX at 5 M was included in the pipette solution, and repetitive pulses of 24 ms at 50 mV were applied at 2 Hz in cells expressing 1-wild-type (C) and 1F1579K (D) mutant channels.
Thus two out of three residues critical for LA binding ( 1-F1579K and 1-N1584K but not 1-Y1586K) display a complete BTXresistant phenotype after the lysine substitution.
Such a suggestion seems to be supported by the result for the mutant 1-F1579K, which also displays a complete BTX-resistant phenotype.
6) and that point mutations at 1-F1579K and 1-N1584K render channels resistant to BTX (Figs.
In summary, we have created several D4 S6 mutants of muscle 1 Na channels that show a complete ( 1-F1579K FIGURE 8 Upper panel illustrates the cut-open view of four domains of the Na channel -subunit.
N1584A
protein
substitution
P15390
true positive
Mutants of 1-F1579K, 1-F1579A, 1-N1584K, 1-N1584A, 1-N1584D, 1Y1586K, 1-Y1586A, and hH1-N1765A were used in this study.
As in brain Na channels for homologous mutations, the point mutation at 1-F1579A reduces LA binding, whereas the point mutation at 1-N1584A drastically enhances LA binding (Wang et al., 1998b).
The 1-F1579A mutant channels are readily modified by BTX, whereas the 1-N1584A channels remain relatively BTX resistant (Fig.
Notice that there is little BTX-induced steady-state current in the 1-N1584A after repetitive pulses, although a slower decaying phase becomes evident in the presence of BTX.
Hence, 1-N1584A is not completely resistant to BTX modification under our assay conditions.
The expression of 1-N1584A is generally poor, and detailed studies of this mutant are difficult (Wang et al., 1998b).
These results suggest that the positive charge brought in by the lysine mutation at the 1-F1579 position may be crucial for the BTX-resistant phenotype, but less so at the 1-N1584 Wang and Wang BTX-Resistant Na channels 3145 FIGURE 4 Superimposed current traces were recorded from cells expressing 1-F1579A (A), 1-N1584A (B), 1-Y1586A (C), and hH1-N1765A (D).
Peak current amplitudes in 1-F1579A, 1-N1584A, and hH1-N1765A were significantly reduced during repetitive pulses.
Expression of 1-N1584A and hH1-N1765A was generally poor in most cells ( 1 nA).
Because 1-N1584A is the only nonlysine mutant that displays a partial BTX-resistant phenotype thus far in this series of mutants, we tested whether the equivalent mutant in the human heart Na channel isoform hH1 (Gellens et al., 1992) is also partially BTX-resistant.
4 D shows that the hH1-N1765A current indeed displays a partially BTXresistant phenotype similar to that of the 1-N1584A cur- rent; a slower decaying phase is again evident after repetitive pulses.
Why 1-N1584A become partially BTX-resistant is unclear.
The numbers of experiments for wild-type, F1579A, N1584A, and Y1586K are 7, 6, 8, and 5, respectively.
Direct BTX bindN1584D 1-N1584A ing studies will be required to confirm this inference.
This phenotype is the opposite of the enhanced LA block in 1-N1584A, possibly due to residuespecific allosteric effects.
Wang and Wang BTX-Resistant Na channels 3149 and 1-N1584K) or a partial BTX-resistant phenotype ( 1N1584A and 1-N1584D).
10354437
full text
C725B
protein
substitution
true negative
Membrane currents were studied using the two-microelectrode voltage clamp technique, with an Oocyte Clamp amplifier (model OC725B; Warner Instrument Corp, MA).
S631E
protein
substitution
true positive
Q12809
The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker.
S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker.
Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E.
To test how changing side chain properties at position 631 could affect the C-type inactivation process in hERG, we mutated S631 to a number of residues: an aromatic residue with a hydroxyl group (S631Y), hydrophobic residues with different sizes (small side chain in S631A and larger side chain in S631V), or hydrophilic residues with a positive (S631K) or a negative (S631E) charge.
If the physicochemical properties of side chain at position 631 required to sustain an efficient C-type inactivation process in the hERG channel were the same as those for T449 in Shaker (see Introduction) (Ogielska et al., 1995; Lopez-Barneo et al., 1993), we expected to see a reduction in the degree of C-type inactivation in S631Y and S631V, but a stronger C-type inactivation in S631K, S631E, and S631A.
3132 Biophysical Journal Volume 76 June 1999 S631E, there was a slow decay phase upon depolarization to 20 mV or more positive voltages, although the overall current amplitude showed an outward rectification.
The slow decay phase of S631E suggests that this mutant has a voltagedependent inactivation process, the rate of which is much slower than that of C-type inactivation in the WT channel.
Again, the degree of C-type inactivation was most prominent in WT, retained (although to a lesser degree) in S631Y and S631A and apparently absent in S631V, S631K, and S631E.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
3, the current traces recorded from S631K, S631E, and (to a lesser degree) S631V showed a time-dependent increase in the inward direction in the voltage range close to the threshold of channel activation.
4): currents were inward during small depolarization steps ( 60 to 20 mV for S631K, 60 to 10 mV for S631E, 60 to 30 mV for S631V).
The positive shift in Erev of S631V, S631K, and S631E suggests that extracellular Na ions may be able to carry charges through these mutant channels in the presence of K ions.
Removing extracellular Na ions did not shift Erev of WT, S631Y, or S631A, but caused a marked negative shift in Erev of S631V, S631K, and S631E.
The degrees of shift in Erev correspond to permeability ratios of Na to K (PNa/PK) of 0.05, 0.09, and 0.12 for S631V, S631K, and S631E, respectively (Table 1).
For each cell, It amplitudes were normalized by the maximum outward It measured by 0 mV (WT), 10 mV (S631Y), 10 mV (S631A), or 60 mV (S631V, S631K, and S631E).
Role of S631 in hERG Channel Function 3133 TABLE 1 Effects of removing extracellular Na ions on the reversal potentials of the wild-type and S631 mutant channels of hERG* [Na]o (mM)# Channel WT S631Y S631A S631V S631K S631E 96 98.2 98.6 94.5 59.4 24.2 12.5 0.7 0.7 1.2 2.8 3.1 1.1 n 12 11 11 14 14 10 0 97.5 95.0 94.3 91.0 66.7 61.0 1.9 1.0 1.5 2.6 3.3 5.1 n 4 2 4 3 3 5 PNa/PK -- -- -- 0.05 0.09 0.12 *Reversal potentials (Erev) were determined from the IV relationships of fully activated currents as shown in Fig.
The permeability ratio of Na to K (PNa/PK) was calculated based on the constant field theory (Hille, 1992), using the shift in mean Erev ( Erev) associated with Na0 removal: PNa/PK 2 10 Erev/58.6 2 /96 (3) The calculation was done for S631V, S631K, and S631E.
In S631V, S631K, and S631E, removing Nao reduced inward currents, but the degree of reduction varied among the three.
Current amplitudes were normalized by the control current (in 96 mM [Na]o) at 50 mV (WT and S631Y), 0 mV (S631A), or 50 mV (S631V, S631K and S631E).
For S631K and S631E, there appeared a shallow component of channel activation in the voltage range positive to 10 or 20 mV.
The half-maximum activation voltage of the major Boltzmann component (the one in the negative voltage range) was shifted in the hyperpolarizing direction relative to that of the WT channel (V0.5 values: S631K 27.4 1.2 and S631E 35.9 1.9 mV, p 0.01 for both mutants versus WT).
3: the rates of activation of S631V, S631K, and S631E appeared faster than that of the WT channel in the negative voltage range.
To verify this observation, we measured and compared the time constants ( ) of activation at various test pulse voltages between WT and S631E.
For S631E that apparently does not C-type inactivate in the negative of activation was obtained by directly voltage range, fitting the current traces during depolarization with a single exponential function.
At 40, 20, and 0 mV, the values of activation for the WT channel were 2261 158 (the fast component), 717 40, and 215 19 ms, and those of S631E were 429 38, 190 37, and 54 8 ms (n 35 each).
Therefore, the S631E mutation greatly accelerated channel activation in the negative voltage range.
Those of S631E and S631K were fit with an empirical double Boltzmann function: Fraction activated A1/ 1 exp V1 Vt /k1 1 A1 / 1 exp V2 Vt /k2 , (2) where A1 is the fraction of the major Boltzmann component in the more negative voltage range, and V1 and k1 are the half-maximum activation voltage and slope factor of this component.
Those that disrupted C-type inactivation (S631V, S631K, and S631E) also lost K selectivity against Na and showed a negative shift in the voltage dependence of activation.
The new results presented here are from other mutations (S631Y, S631K, S631E, and, in particular, S631C).
Mutations that disrupted C-type inactivation (S631V, S631K, and S631E) also reduced the channels' K selectivity (conducting Na ions in the presence of K) and altered the voltage dependence of activation (causing a hyperpolarizing shift in V0.5 of activation, along with an appearance of a shallow component of channel activation in the positive voltage range).
S631C channels with thiol groups modified by an oxidizing agent (H2O2) or by MTSET or MTSES displayed a mutant behavior similar to that described above for S631V, S631K, and S631E.
However, this tight linkage raises a concern: is it possible that the lack of C-type inactivation in these mutants was due to Na ions binding within the pore that hindered C-type inactivation, similar to the effects elevating [K]o on the WT hERG channel (Wang et al., 1996; Sanguinetti et al., 1995)? This is not likely, because a total removal of extracellular Na ions did not restore C-type inactivation in S631V, S631K, S631E, or S631C (H2O2-treated) (Figs.
lectivity accompanying the loss of C-type inactivation seen in S631K and S631E of hERG is not seen in T449K or T449E of Shaker, again pointing to differences in the outer mouth structure between these two channels (Molina et al., 1997; Ogielska et al., 1995; Lopez-Barneo et al., 1993).
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
T449V
protein
substitution
true negative
Putting a bulky hydrophobic residue (T449V) or an aromatic residue (T449Y) at this position hinders C-type inactivation, whereas a hydrophilic residue (T449K or T449E) can greatly facilitate C-type inactivation.
Therefore, only the S631V mutation had the same effect on hERG as that of T449V on Shaker (hindering C-type inactivation).
No such effects were noted for the T449V, T449K, or T449E mutation of the Shaker channel (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
The importance of hydrogen bonds between tryptophan and tyrosine residues in the function of this "molecular spring" is supported by the observations that if one of the tryptophans is mutated to phenylalanine (W434F) (Yang et al., 1997) or if the tyrosine is mutated to valine (F445V) (Heginbotham et al., 1994), so that the hydrogen bonds are interrupted, the channel tends to be persistently C-type inactivated, as if removing the hydrogen bonds and weak- Comparison with equivalent mutations of T449 in the Shaker channel When comparing the effects of S631 mutations with those of T449 mutations in the Shaker channel, only S631V showed an effect similar to that of T449V: C-type inactivation was abolished in both (Ogielska et al., 1995; LopezBarneo et al., 1993).
T449A
protein
substitution
true negative
A small hydrophobic residue here (T449A) can also enhance the rate of C-type inactivation.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
F445V
protein
substitution
true negative
The importance of hydrogen bonds between tryptophan and tyrosine residues in the function of this "molecular spring" is supported by the observations that if one of the tryptophans is mutated to phenylalanine (W434F) (Yang et al., 1997) or if the tyrosine is mutated to valine (F445V) (Heginbotham et al., 1994), so that the hydrogen bonds are interrupted, the channel tends to be persistently C-type inactivated, as if removing the hydrogen bonds and weak- Comparison with equivalent mutations of T449 in the Shaker channel When comparing the effects of S631 mutations with those of T449 mutations in the Shaker channel, only S631V showed an effect similar to that of T449V: C-type inactivation was abolished in both (Ogielska et al., 1995; LopezBarneo et al., 1993).
S631C
protein
substitution
true positive
Q12809
S631C behaved like the wild-type channel when the thiol groups were in the reduced state.
To further test the linkage between side-chain properties at position 631 and channel function, we mutated S631 to cysteine (S631C).
We then studied how modifying the thiol groups at position 631 could affect the gating function and current amplitude of the S631C mutant.
Effects of DTT or H2O2 on the I-V relationship and voltage dependence of activation of S631C mutant The left panels of Fig.
8, A and B, show current traces and the I-V relationship of S631C recorded from an oocyte that had been treated with 5 mM DTT for 15 min.
Effects of MTSET and MTSES on the I-V relationship and voltage dependence of activation of S631C mutant We further examined how modifying the thiol group at 631 by MTS reagents could alter the S631C channel properties.
9, AC, illustrates the effects of MTSET on S631C.
This caused a switch of S631C phenotype from "WT-like" to a mutant behavior that was similar, although not identical, to that induced by H2O2.
9, DF, illustrates the effects of MTSES on S631C.
FIGURE 8 Redox state of thiol groups influences the phenotype of hERG S631C.
(A) Effects of H2O2 (0.1%) on hERG S631C recorded from a DTT-treated oocyte.
(B) Effects of removing extracellular Na ions on hERG S631C recorded from a DTT-treated oocyte (left) or from an H2O2-treated oocyte (right).
(CE) Comparison of hERG S631C channel properties with thiol groups in the reduced state (DTT-treated, E) or in oxidized states (H2O2treated, OE).
(C) The current-voltage relationships of S631C currents recorded during depolarization pulses (It).
(D) The current-voltage relationships of fully activated hERG S631C currents (Ifully activated).
8, CE, summarizes the properties of S631C in oocytes treated with DTT or with H2O2.
In H2O2-treated oocytes (thiol groups in disulfide bonds or in higher oxidized states) Switch of S631C phenotype by repetitive depolarization pulses S631C in oocytes that had not been treated with DTT showed a behavior intermediate between "WT-like" and mutant forms.
Role of S631 in hERG Channel Function 3137 FIGURE 9 Effects of thiol-specific methanethiosulfonate reagents on hERG S631C.
(DF) Effects of MTSES on hERG S631C.
This behavior indicated that without DTT treatment the S631C channels might exist as a mixed population of "WT-like" and mutant forms.
The new results presented here are from other mutations (S631Y, S631K, S631E, and, in particular, S631C).
FIGURE 10 Switch of hERG S631C channel phenotype induced by repetitive depolarization pulses (28 pulses to 40 mV for 1 s each) and its reversal by a thiol-reducing agent (5 mM DTT).
A more direct linkage between 631 sidechain properties and channel functions was seen in the S631C mutant.
S631C channels that had thiol groups in the reduced state (free thiol groups) behaved like the WT channel.
S631C channels with thiol groups modified by an oxidizing agent (H2O2) or by MTSET or MTSES displayed a mutant behavior similar to that described above for S631V, S631K, and S631E.
However, this tight linkage raises a concern: is it possible that the lack of C-type inactivation in these mutants was due to Na ions binding within the pore that hindered C-type inactivation, similar to the effects elevating [K]o on the WT hERG channel (Wang et al., 1996; Sanguinetti et al., 1995)? This is not likely, because a total removal of extracellular Na ions did not restore C-type inactivation in S631V, S631K, S631E, or S631C (H2O2-treated) (Figs.
First, S631C of hERG could form disulfide bonds spontaneously (Fig.
When cysteine residues occupy position 631 in the four subunits (S631C), membrane depolarization and thus C- type inactivation will bring them close enough to each other to form disulfide bonds.
T449Y
protein
substitution
true negative
Putting a bulky hydrophobic residue (T449V) or an aromatic residue (T449Y) at this position hinders C-type inactivation, whereas a hydrophilic residue (T449K or T449E) can greatly facilitate C-type inactivation.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
This is in sharp contrast to the situation in the Shaker channel: T449Y greatly increases the channel's sensitivity to external TEA by allowing interactions between the blocker's positive charge and electrons of tyrosine residues in the outer mouth region (Heginbotham and MacKinnon, 1992).
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
S641E
protein
substitution
true positive
Q12809
Furthermore, when glutamate or lysine residues occupy this position in the four subunits (S641E or S631K), the repulsion between like charges will limit the degree of freedom of side chain and backbone movements, leading to a disruption of C-type inactivation and K selectivity.
S631K
protein
substitution
true positive
Q12809
The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker.
S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker.
Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E.
To test how changing side chain properties at position 631 could affect the C-type inactivation process in hERG, we mutated S631 to a number of residues: an aromatic residue with a hydroxyl group (S631Y), hydrophobic residues with different sizes (small side chain in S631A and larger side chain in S631V), or hydrophilic residues with a positive (S631K) or a negative (S631E) charge.
If the physicochemical properties of side chain at position 631 required to sustain an efficient C-type inactivation process in the hERG channel were the same as those for T449 in Shaker (see Introduction) (Ogielska et al., 1995; Lopez-Barneo et al., 1993), we expected to see a reduction in the degree of C-type inactivation in S631Y and S631V, but a stronger C-type inactivation in S631K, S631E, and S631A.
In S631V and S631K, the degree of C-type inactivation was further reduced or abolished, so that current amplitudes continued to increase up to 60 mV.
Again, the degree of C-type inactivation was most prominent in WT, retained (although to a lesser degree) in S631Y and S631A and apparently absent in S631V, S631K, and S631E.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
3, the current traces recorded from S631K, S631E, and (to a lesser degree) S631V showed a time-dependent increase in the inward direction in the voltage range close to the threshold of channel activation.
4): currents were inward during small depolarization steps ( 60 to 20 mV for S631K, 60 to 10 mV for S631E, 60 to 30 mV for S631V).
Azimilide blocks the WT and S631 mutant channels with an IC50 ranging from 3 M (S631K) to 14 M (S631Y) (n 4 9 each, measured at 20 mV).
The positive shift in Erev of S631V, S631K, and S631E suggests that extracellular Na ions may be able to carry charges through these mutant channels in the presence of K ions.
Removing extracellular Na ions did not shift Erev of WT, S631Y, or S631A, but caused a marked negative shift in Erev of S631V, S631K, and S631E.
The degrees of shift in Erev correspond to permeability ratios of Na to K (PNa/PK) of 0.05, 0.09, and 0.12 for S631V, S631K, and S631E, respectively (Table 1).
For each cell, It amplitudes were normalized by the maximum outward It measured by 0 mV (WT), 10 mV (S631Y), 10 mV (S631A), or 60 mV (S631V, S631K, and S631E).
Role of S631 in hERG Channel Function 3133 TABLE 1 Effects of removing extracellular Na ions on the reversal potentials of the wild-type and S631 mutant channels of hERG* [Na]o (mM)# Channel WT S631Y S631A S631V S631K S631E 96 98.2 98.6 94.5 59.4 24.2 12.5 0.7 0.7 1.2 2.8 3.1 1.1 n 12 11 11 14 14 10 0 97.5 95.0 94.3 91.0 66.7 61.0 1.9 1.0 1.5 2.6 3.3 5.1 n 4 2 4 3 3 5 PNa/PK -- -- -- 0.05 0.09 0.12 *Reversal potentials (Erev) were determined from the IV relationships of fully activated currents as shown in Fig.
The permeability ratio of Na to K (PNa/PK) was calculated based on the constant field theory (Hille, 1992), using the shift in mean Erev ( Erev) associated with Na0 removal: PNa/PK 2 10 Erev/58.6 2 /96 (3) The calculation was done for S631V, S631K, and S631E.
In S631V, S631K, and S631E, removing Nao reduced inward currents, but the degree of reduction varied among the three.
Current amplitudes were normalized by the control current (in 96 mM [Na]o) at 50 mV (WT and S631Y), 0 mV (S631A), or 50 mV (S631V, S631K and S631E).
For S631K and S631E, there appeared a shallow component of channel activation in the voltage range positive to 10 or 20 mV.
The half-maximum activation voltage of the major Boltzmann component (the one in the negative voltage range) was shifted in the hyperpolarizing direction relative to that of the WT channel (V0.5 values: S631K 27.4 1.2 and S631E 35.9 1.9 mV, p 0.01 for both mutants versus WT).
3: the rates of activation of S631V, S631K, and S631E appeared faster than that of the WT channel in the negative voltage range.
Those of S631E and S631K were fit with an empirical double Boltzmann function: Fraction activated A1/ 1 exp V1 Vt /k1 1 A1 / 1 exp V2 Vt /k2 , (2) where A1 is the fraction of the major Boltzmann component in the more negative voltage range, and V1 and k1 are the half-maximum activation voltage and slope factor of this component.
Those that disrupted C-type inactivation (S631V, S631K, and S631E) also lost K selectivity against Na and showed a negative shift in the voltage dependence of activation.
The new results presented here are from other mutations (S631Y, S631K, S631E, and, in particular, S631C).
Mutations that disrupted C-type inactivation (S631V, S631K, and S631E) also reduced the channels' K selectivity (conducting Na ions in the presence of K) and altered the voltage dependence of activation (causing a hyperpolarizing shift in V0.5 of activation, along with an appearance of a shallow component of channel activation in the positive voltage range).
S631C channels with thiol groups modified by an oxidizing agent (H2O2) or by MTSET or MTSES displayed a mutant behavior similar to that described above for S631V, S631K, and S631E.
However, this tight linkage raises a concern: is it possible that the lack of C-type inactivation in these mutants was due to Na ions binding within the pore that hindered C-type inactivation, similar to the effects elevating [K]o on the WT hERG channel (Wang et al., 1996; Sanguinetti et al., 1995)? This is not likely, because a total removal of extracellular Na ions did not restore C-type inactivation in S631V, S631K, S631E, or S631C (H2O2-treated) (Figs.
lectivity accompanying the loss of C-type inactivation seen in S631K and S631E of hERG is not seen in T449K or T449E of Shaker, again pointing to differences in the outer mouth structure between these two channels (Molina et al., 1997; Ogielska et al., 1995; Lopez-Barneo et al., 1993).
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
Furthermore, when glutamate or lysine residues occupy this position in the four subunits (S641E or S631K), the repulsion between like charges will limit the degree of freedom of side chain and backbone movements, leading to a disruption of C-type inactivation and K selectivity.
T449C
protein
substitution
true negative
10), whereas T449C of Shaker cannot (Liu et al., 1996).
However, these residues still have sufficient distance between them such that when cysteine residues occupy this position (T449C) they cannot form disulfide bonds with each other.
S631V
protein
substitution
true positive
Q12809
To test how changing side chain properties at position 631 could affect the C-type inactivation process in hERG, we mutated S631 to a number of residues: an aromatic residue with a hydroxyl group (S631Y), hydrophobic residues with different sizes (small side chain in S631A and larger side chain in S631V), or hydrophilic residues with a positive (S631K) or a negative (S631E) charge.
If the physicochemical properties of side chain at position 631 required to sustain an efficient C-type inactivation process in the hERG channel were the same as those for T449 in Shaker (see Introduction) (Ogielska et al., 1995; Lopez-Barneo et al., 1993), we expected to see a reduction in the degree of C-type inactivation in S631Y and S631V, but a stronger C-type inactivation in S631K, S631E, and S631A.
In S631V and S631K, the degree of C-type inactivation was further reduced or abolished, so that current amplitudes continued to increase up to 60 mV.
Again, the degree of C-type inactivation was most prominent in WT, retained (although to a lesser degree) in S631Y and S631A and apparently absent in S631V, S631K, and S631E.
Therefore, only the S631V mutation had the same effect on hERG as that of T449V on Shaker (hindering C-type inactivation).
3, the current traces recorded from S631K, S631E, and (to a lesser degree) S631V showed a time-dependent increase in the inward direction in the voltage range close to the threshold of channel activation.
4): currents were inward during small depolarization steps ( 60 to 20 mV for S631K, 60 to 10 mV for S631E, 60 to 30 mV for S631V).
The positive shift in Erev of S631V, S631K, and S631E suggests that extracellular Na ions may be able to carry charges through these mutant channels in the presence of K ions.
Removing extracellular Na ions did not shift Erev of WT, S631Y, or S631A, but caused a marked negative shift in Erev of S631V, S631K, and S631E.
The degrees of shift in Erev correspond to permeability ratios of Na to K (PNa/PK) of 0.05, 0.09, and 0.12 for S631V, S631K, and S631E, respectively (Table 1).
For each cell, It amplitudes were normalized by the maximum outward It measured by 0 mV (WT), 10 mV (S631Y), 10 mV (S631A), or 60 mV (S631V, S631K, and S631E).
Role of S631 in hERG Channel Function 3133 TABLE 1 Effects of removing extracellular Na ions on the reversal potentials of the wild-type and S631 mutant channels of hERG* [Na]o (mM)# Channel WT S631Y S631A S631V S631K S631E 96 98.2 98.6 94.5 59.4 24.2 12.5 0.7 0.7 1.2 2.8 3.1 1.1 n 12 11 11 14 14 10 0 97.5 95.0 94.3 91.0 66.7 61.0 1.9 1.0 1.5 2.6 3.3 5.1 n 4 2 4 3 3 5 PNa/PK -- -- -- 0.05 0.09 0.12 *Reversal potentials (Erev) were determined from the IV relationships of fully activated currents as shown in Fig.
The permeability ratio of Na to K (PNa/PK) was calculated based on the constant field theory (Hille, 1992), using the shift in mean Erev ( Erev) associated with Na0 removal: PNa/PK 2 10 Erev/58.6 2 /96 (3) The calculation was done for S631V, S631K, and S631E.
In S631V, S631K, and S631E, removing Nao reduced inward currents, but the degree of reduction varied among the three.
Current amplitudes were normalized by the control current (in 96 mM [Na]o) at 50 mV (WT and S631Y), 0 mV (S631A), or 50 mV (S631V, S631K and S631E).
The activation curve of S631V was shifted in the negative direction relative to that of the WT channel (V0.5 21.5 2.0 mV, p 0.01).
3: the rates of activation of S631V, S631K, and S631E appeared faster than that of the WT channel in the negative voltage range.
Activation curves of WT, S631Y, S631A, and S631V were fit with a simple Boltzmann function (Eq.
Those that disrupted C-type inactivation (S631V, S631K, and S631E) also lost K selectivity against Na and showed a negative shift in the voltage dependence of activation.
Our observations that C-type inactivation was attenuated in S631A and abolished in S631V are similar to those of others.
Mutations that disrupted C-type inactivation (S631V, S631K, and S631E) also reduced the channels' K selectivity (conducting Na ions in the presence of K) and altered the voltage dependence of activation (causing a hyperpolarizing shift in V0.5 of activation, along with an appearance of a shallow component of channel activation in the positive voltage range).
S631C channels with thiol groups modified by an oxidizing agent (H2O2) or by MTSET or MTSES displayed a mutant behavior similar to that described above for S631V, S631K, and S631E.
However, this tight linkage raises a concern: is it possible that the lack of C-type inactivation in these mutants was due to Na ions binding within the pore that hindered C-type inactivation, similar to the effects elevating [K]o on the WT hERG channel (Wang et al., 1996; Sanguinetti et al., 1995)? This is not likely, because a total removal of extracellular Na ions did not restore C-type inactivation in S631V, S631K, S631E, or S631C (H2O2-treated) (Figs.
The importance of hydrogen bonds between tryptophan and tyrosine residues in the function of this "molecular spring" is supported by the observations that if one of the tryptophans is mutated to phenylalanine (W434F) (Yang et al., 1997) or if the tyrosine is mutated to valine (F445V) (Heginbotham et al., 1994), so that the hydrogen bonds are interrupted, the channel tends to be persistently C-type inactivated, as if removing the hydrogen bonds and weak- Comparison with equivalent mutations of T449 in the Shaker channel When comparing the effects of S631 mutations with those of T449 mutations in the Shaker channel, only S631V showed an effect similar to that of T449V: C-type inactivation was abolished in both (Ogielska et al., 1995; LopezBarneo et al., 1993).
H2N
protein
substitution
true negative
MTSET added a positively charged moiety to a thiol group: -CH2-CH2N (CH3)3.
S631A
protein
substitution
true positive
Q12809
To test how changing side chain properties at position 631 could affect the C-type inactivation process in hERG, we mutated S631 to a number of residues: an aromatic residue with a hydroxyl group (S631Y), hydrophobic residues with different sizes (small side chain in S631A and larger side chain in S631V), or hydrophilic residues with a positive (S631K) or a negative (S631E) charge.
If the physicochemical properties of side chain at position 631 required to sustain an efficient C-type inactivation process in the hERG channel were the same as those for T449 in Shaker (see Introduction) (Ogielska et al., 1995; Lopez-Barneo et al., 1993), we expected to see a reduction in the degree of C-type inactivation in S631Y and S631V, but a stronger C-type inactivation in S631K, S631E, and S631A.
In S631Y and S631A, the C-type inactivation process was retained, as evidenced by the decrease in outward currents during depolarization pulses positive to 10 mV (S631Y) or 10 mV (S631A).
Again, the degree of C-type inactivation was most prominent in WT, retained (although to a lesser degree) in S631Y and S631A and apparently absent in S631V, S631K, and S631E.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
Removing extracellular Na ions did not shift Erev of WT, S631Y, or S631A, but caused a marked negative shift in Erev of S631V, S631K, and S631E.
For each cell, It amplitudes were normalized by the maximum outward It measured by 0 mV (WT), 10 mV (S631Y), 10 mV (S631A), or 60 mV (S631V, S631K, and S631E).
Role of S631 in hERG Channel Function 3133 TABLE 1 Effects of removing extracellular Na ions on the reversal potentials of the wild-type and S631 mutant channels of hERG* [Na]o (mM)# Channel WT S631Y S631A S631V S631K S631E 96 98.2 98.6 94.5 59.4 24.2 12.5 0.7 0.7 1.2 2.8 3.1 1.1 n 12 11 11 14 14 10 0 97.5 95.0 94.3 91.0 66.7 61.0 1.9 1.0 1.5 2.6 3.3 5.1 n 4 2 4 3 3 5 PNa/PK -- -- -- 0.05 0.09 0.12 *Reversal potentials (Erev) were determined from the IV relationships of fully activated currents as shown in Fig.
For WT, S631Y, and S631A there was no measurable Na permeability (no negative shift in Erev when Na0 was removed).
In particular, in the voltage range negative to Erev, removing Nao reduced inward currents through the WT and S631Y channels, but had the opposite effect on S631A.
Current amplitudes were normalized by the control current (in 96 mM [Na]o) at 50 mV (WT and S631Y), 0 mV (S631A), or 50 mV (S631V, S631K and S631E).
For S631Y and S631A, the activation curves were largely superimposable on that of the WT channel (V0.5 values: WT 13.4 1.1 mV, S631Y 17.5 2.5 mV, S631A 14.5 1.9 mV, p 0.05 for both mutants versus WT).
Activation curves of WT, S631Y, S631A, and S631V were fit with a simple Boltzmann function (Eq.
Role of S631 in hERG Channel Function 3135 retained the C-type inactivation process (S631Y and S631A) maintained a high K selectivity.
Our observations that C-type inactivation was attenuated in S631A and abolished in S631V are similar to those of others.
Mutations that retained C-type inactivation (S631Y and S631A) maintained a high K selectivity and the same voltage dependence of activation as the WT channel.
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
T449E
protein
substitution
true negative
The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker.
S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker.
Putting a bulky hydrophobic residue (T449V) or an aromatic residue (T449Y) at this position hinders C-type inactivation, whereas a hydrophilic residue (T449K or T449E) can greatly facilitate C-type inactivation.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
No such effects were noted for the T449V, T449K, or T449E mutation of the Shaker channel (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
lectivity accompanying the loss of C-type inactivation seen in S631K and S631E of hERG is not seen in T449K or T449E of Shaker, again pointing to differences in the outer mouth structure between these two channels (Molina et al., 1997; Ogielska et al., 1995; Lopez-Barneo et al., 1993).
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
When glutamate or lysine residues occupy this position (T449E or T449K), there is no repulsion between the like charges from different subunits.
T449K
protein
substitution
true negative
The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker.
S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker.
Putting a bulky hydrophobic residue (T449V) or an aromatic residue (T449Y) at this position hinders C-type inactivation, whereas a hydrophilic residue (T449K or T449E) can greatly facilitate C-type inactivation.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
No such effects were noted for the T449V, T449K, or T449E mutation of the Shaker channel (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
lectivity accompanying the loss of C-type inactivation seen in S631K and S631E of hERG is not seen in T449K or T449E of Shaker, again pointing to differences in the outer mouth structure between these two channels (Molina et al., 1997; Ogielska et al., 1995; Lopez-Barneo et al., 1993).
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
When glutamate or lysine residues occupy this position (T449E or T449K), there is no repulsion between the like charges from different subunits.
M448C
protein
substitution
true negative
However, M448C, which is one residue deeper into the pore and thus is in a more confined environment, can form disulfide bonds with other M448C across subunits (Liu et al., 1996).
S631Y
protein
substitution
true positive
Q12809
To test how changing side chain properties at position 631 could affect the C-type inactivation process in hERG, we mutated S631 to a number of residues: an aromatic residue with a hydroxyl group (S631Y), hydrophobic residues with different sizes (small side chain in S631A and larger side chain in S631V), or hydrophilic residues with a positive (S631K) or a negative (S631E) charge.
If the physicochemical properties of side chain at position 631 required to sustain an efficient C-type inactivation process in the hERG channel were the same as those for T449 in Shaker (see Introduction) (Ogielska et al., 1995; Lopez-Barneo et al., 1993), we expected to see a reduction in the degree of C-type inactivation in S631Y and S631V, but a stronger C-type inactivation in S631K, S631E, and S631A.
In S631Y and S631A, the C-type inactivation process was retained, as evidenced by the decrease in outward currents during depolarization pulses positive to 10 mV (S631Y) or 10 mV (S631A).
Again, the degree of C-type inactivation was most prominent in WT, retained (although to a lesser degree) in S631Y and S631A and apparently absent in S631V, S631K, and S631E.
The other mutations either induced different effects (S631Y sustained C-type inactivation in hERG, whereas T449Y hindered C-type inactivation in Shaker) or had opposite effects (S631A, S631K, and S631E reduced or abolished Ctype inactivation in hERG, whereas T449A, T449K, and T449E enhanced C-type inactivation in Shaker).
Azimilide blocks the WT and S631 mutant channels with an IC50 ranging from 3 M (S631K) to 14 M (S631Y) (n 4 9 each, measured at 20 mV).
Removing extracellular Na ions did not shift Erev of WT, S631Y, or S631A, but caused a marked negative shift in Erev of S631V, S631K, and S631E.
For each cell, It amplitudes were normalized by the maximum outward It measured by 0 mV (WT), 10 mV (S631Y), 10 mV (S631A), or 60 mV (S631V, S631K, and S631E).
Role of S631 in hERG Channel Function 3133 TABLE 1 Effects of removing extracellular Na ions on the reversal potentials of the wild-type and S631 mutant channels of hERG* [Na]o (mM)# Channel WT S631Y S631A S631V S631K S631E 96 98.2 98.6 94.5 59.4 24.2 12.5 0.7 0.7 1.2 2.8 3.1 1.1 n 12 11 11 14 14 10 0 97.5 95.0 94.3 91.0 66.7 61.0 1.9 1.0 1.5 2.6 3.3 5.1 n 4 2 4 3 3 5 PNa/PK -- -- -- 0.05 0.09 0.12 *Reversal potentials (Erev) were determined from the IV relationships of fully activated currents as shown in Fig.
For WT, S631Y, and S631A there was no measurable Na permeability (no negative shift in Erev when Na0 was removed).
In particular, in the voltage range negative to Erev, removing Nao reduced inward currents through the WT and S631Y channels, but had the opposite effect on S631A.
These observations suggest that (other than S631Y) S631 FIGURE 5 Effects of removing external Na ions on the current-voltage relationships of Ifully activated and the reversal potentials of hERG WT and S631 mutants.
Current amplitudes were normalized by the control current (in 96 mM [Na]o) at 50 mV (WT and S631Y), 0 mV (S631A), or 50 mV (S631V, S631K and S631E).
Data were averaged from three to five experiments for each channel (except S631Y, n 2).
For S631Y and S631A, the activation curves were largely superimposable on that of the WT channel (V0.5 values: WT 13.4 1.1 mV, S631Y 17.5 2.5 mV, S631A 14.5 1.9 mV, p 0.05 for both mutants versus WT).
Activation curves of WT, S631Y, S631A, and S631V were fit with a simple Boltzmann function (Eq.
Role of S631 in hERG Channel Function 3135 retained the C-type inactivation process (S631Y and S631A) maintained a high K selectivity.
The new results presented here are from other mutations (S631Y, S631K, S631E, and, in particular, S631C).
Mutations that retained C-type inactivation (S631Y and S631A) maintained a high K selectivity and the same voltage dependence of activation as the WT channel.
Second, the S631Y mutation did not enhance the channel's sensitivity to extracellular TEA (data not shown), suggesting that this extracellular blocker cannot reach the side chain at position 631.
The others were either different (S631Y retained C-type inactivation in hERG, whereas T449Y abolishes C-type in Shaker), or opposite (S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E greatly facilitated C-type inactivation in Shaker; S631A retained C-type inactivation but to a lesser degree than that of WT hERG, whereas T449A enhanced C-type inactivation in Shaker) (Ogielska et al., 1995; Lopez-Barneo et al., 1993).
W434F
protein
substitution
true negative
The importance of hydrogen bonds between tryptophan and tyrosine residues in the function of this "molecular spring" is supported by the observations that if one of the tryptophans is mutated to phenylalanine (W434F) (Yang et al., 1997) or if the tyrosine is mutated to valine (F445V) (Heginbotham et al., 1994), so that the hydrogen bonds are interrupted, the channel tends to be persistently C-type inactivated, as if removing the hydrogen bonds and weak- Comparison with equivalent mutations of T449 in the Shaker channel When comparing the effects of S631 mutations with those of T449 mutations in the Shaker channel, only S631V showed an effect similar to that of T449V: C-type inactivation was abolished in both (Ogielska et al., 1995; LopezBarneo et al., 1993).
10354435
full text
S942A
protein
substitution
true negative
Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane.
Therefore, oocytes were microinjected with either cRNA transcribed in vitro from cDNA encoding variant A1/C2/E1/G3/I0 of dSlo (wild-type channel) (Adelman et al., 1992) or with cRNA in which a putative PKA phosphorylation site was eliminated by replacing serine at position 942 with alanine (S942A channel), as described previously (Esguerra et al., 1994).
Unless otherwise indicated, experiments were performed on patches containing a single S942A channel, determined by extended recordings at levels of Ca2 that would be exi pected to readily activate BK channels.
Recordings were obtained from a total of 53 excised patches (39 S942A and 14 wild-type), each containing a single dSlo channel.
The results presented in this paper were obtained from the remaining 14 dSlo channels (12 S942A and 2 wild-type).
Although most of the channels studied were S942A, there was no obvious indication that wild-type channels were less stable than the mutant (Silberberg et al., 1996) or had different kinetics.
Four of the dSlo channels were S942A and one was wild-type (channel H01, ).
12228229
full text
S32P
protein
substitution
true negative
Additionally, the site-directed mutation of serine 32 to proline (S32P) in BFV 6K protein resulted in altered ionic selectivity of the channel.
Site-directed Mutagenesis--To create a mutation from serine to proline at position 32 of BFV 6K (named BFV 6K S32P) recombinant PCR was used (26).
However, in comparison to RRV 6K, the BFV 46928 Alphavirus 6K Proteins Form Ion Channels TABLE I Reversal potentials (mV) for 60 mM NaCl (trans) vs: Channel species NaCl KCl CaCl2 BFV 6K (wt) BFV 6K S32P RRV 6K (wt) 48.3 46.9 49.1 1.9 (n 1.6 (n 0.7 (n 9) 13) 12) 31.7 37.2 33.4 Permeability ratios 1.4 (n 0.7 (n 1.0 (n 8) 5) 11) 4.3 12.7 22.5 1.2 (n 0.9 (n 2.5 (n 6) 9) 7) Bhannel C species PNa /PC1 PNa /PK PNa /PCa2 FV 6K (wt) BFV 6K S32P RRV 6K (wt) 14.6 12.2 16.3 4.3 2.7 1.7 1.3 1.0 1.3 0.1 0.03 0.05 6.2 3.6 2.6 0.1 0.2 0.4 FIG.
A loss of Na selectivity is indicated by a reduction of PNa /PK from 1.3 in wild-type BFV 6K, to 1.0 in BFV 6K (S32P) (p 0.01).
Secondly, a single nucleotide substitution (S32P) was sufficient to modify the calcium permeability of BFV 6K protein.
In choosing to make the mutation S32P in the BFV 6K protein, we sought to introduce a major structural change within the transmembrane domain as well as removing a potential cation-interacting residue.
BFV 6K channels containing the S32P mutation were less cation-selective than wild-type channels, and the PNa / PCa2 ratio was decreased (see Table I).
15024024
full text
N295Q
protein
substitution
true positive
Q29441
In contrast, every one of the non-functional S4 mutants we tested, whether mutated in charged (R296Q) or neutral (N295Q) amino acids, where not at the plasma membrane, but localized in spatially restricted compartments within the cytoplasm of the cell.
Proteins that did not yield functional channels (R296Q and N295Q) are expressed in compartments within the cell cytoplasm.
Proteins that did not yield functional channels (R296Q and N295Q) are localized at the endoplasmic reticulum.
Proteins that did not yield functional channels (R296Q and N295Q) also are not localized at the Golgi apparatus.
R296Q
protein
substitution
true positive
Q29441
A point mutation in the S4 motif that changed R296Q caused failure of functional channel expression (panel B).
Functional Consequence of Various Point Mutations in S4 --We transformed cells with bCNGA3 cDNA modified to mutate R296Q, this is the second Arg in the S4 motif of the 22646 S4 and Cellular Protein Processing of CNG Ion Channels TABLE I Electrophysiological characterization of the consequence of point mutations in S4 in cone bCNGA3 ion channels and its tagged derivative bCNGA3-C-HA Position COOH-tagged Mutation I hold at 40 mV I flash at pA 75 mV Range Responsive/total cellsa wt wt Arg293 Arg293 Phe294 Asn295 Arg296 Arg296 Arg296 Arg296 Leu297 Lys299 Lys299 Leu300 Arg302 Arg302 Arg302 Leu303 a HA HA Gln Gln Leu, Ala Gln Gln GlnCysb Cys Ala Gln Gln Phe Trpb Trp Gln Ala HA HA HA HA HA 6.8 13.8 11.3 5.0 12.1 10.7 9.6 6.0 10.6 3.0 7.4 30 11.5 7.8 9.6 7.0 9.6 6 6.4 13.9 10.1 3.5 11.1 8.4 9.6 0.8 4.5 1.0 9.4 22 16.9 6.1 5.5 2.6 7.4 74.0 84.6 0 0 119.3 0 0 0 0 0 0 0 0 54.4 0 0 0 103 50.5 94 143.4 36 to 15 to 28 to 170 280 400 26.9 16 to 90 61.8 34 to 177 6/7 11/11 0/4 0/4 6/7 0/7 0/10 0/4 0/3 0/5 0/8 0/2 0/4 5/6 0/1 0/5 0/6 5/5 Counting cells transformed with wt bCNGA3 and tested at voltages other than flash release of pCPT-cGMP.
In contrast, every one of the non-functional S4 mutants we tested, whether mutated in charged (R296Q) or neutral (N295Q) amino acids, where not at the plasma membrane, but localized in spatially restricted compartments within the cytoplasm of the cell.
Proteins that did not yield functional channels (R296Q and N295Q) are expressed in compartments within the cell cytoplasm.
every mutant class tested, we list the mean holding current at 40 mV and the number of cells tested: 1) charged to neutral amino acids, R296Q (Im 0.5 pA, n 2); 2) neutral to neutral amino acid, L297A (Im 2.3 pA, n 4); and 3) human natural mutation: R302W (Im 10 pA, n 3).
Proteins that did not yield functional channels (R296Q and N295Q) are localized at the endoplasmic reticulum.
Proteins that did not yield functional channels (R296Q and N295Q) also are not localized at the Golgi apparatus.
F294L
protein
substitution
true positive
Q29441
These include wt, unglycosylated (gly( )), or functional S4 mutants (F294L).
All channel proteins that yielded functional channels (wt, gly( ), and F294L) are localized to the plasma membrane.
The functional channels, wt, unglycosylated (gly( )), or S4 mutant F294L are not localized in the endoplasmic reticulum.
Every one of the functional channels, wt, unglycosylated (gly( )), or S4 mutant F294L, are not localized in the Golgi.
All channel proteins that yielded functional channels (wt, gly( ), and F294L) are localized at the plasma membrane and not the endoplasmic reticulum.
All channel proteins that yielded functional channels (wt, gly( ), and F294L) are not localized at the Golgi.
L297A
protein
substitution
true positive
Q29441
every mutant class tested, we list the mean holding current at 40 mV and the number of cells tested: 1) charged to neutral amino acids, R296Q (Im 0.5 pA, n 2); 2) neutral to neutral amino acid, L297A (Im 2.3 pA, n 4); and 3) human natural mutation: R302W (Im 10 pA, n 3).
R302Q
protein
substitution
true positive
Q29441
Image mutation
Q296R
protein
substitution
true negative
Reversion of the mutation in the cDNA back to the wt sequence, Q296R, restored normal functional channel expression.
revertant Q296R cDNA, the holding current at 40 mV was 5.6 3.7 pA and peak uncaging flash-generated current at 75 mV was 59.3 36.8 pA (range 20 to 96 pA, n 3).
Typo
R296C
protein
substitution
true positive
Q29441
We measured the electrophysiological properties and channel protein expression pattern in cells transfected with R296C and R302W.
The characteristics of mutants R296C and R302W are indistinguishable.
R302W
protein
substitution
true positive
Q29441
We measured the electrophysiological properties and channel protein expression pattern in cells transfected with R296C and R302W.
The characteristics of mutants R296C and R302W are indistinguishable.
every mutant class tested, we list the mean holding current at 40 mV and the number of cells tested: 1) charged to neutral amino acids, R296Q (Im 0.5 pA, n 2); 2) neutral to neutral amino acid, L297A (Im 2.3 pA, n 4); and 3) human natural mutation: R302W (Im 10 pA, n 3).
10970898
full text
9575283
full text
T449K
protein
substitution
P08510
true positive
These characteristics were particularly apparent in mutant T449K which even in high [K] has a non-linear instantaneous current--voltage relationship with marked saturation of the inward current recorded at negative membrane potentials.
Single amino acid mutation (T449K) with differential effects on closing and C-type inactivation A, slowing of C-type inactivation time course in the T449K channels by external K.
509.2 Channel 1 type at 30 m K at 70 m K at 140 m K (ms) (ms) (ms) T449T (D447D) 26 08 (8) 41 06 (5) T449E 27 05 (5) 32 04 (3) T449K 40 07 (6) 42 06 (4) 43 (1) D447E 07 016 (6) 09 027 (4) 14 (1) was calculated from eqn (1).
An even more marked separation between the changes of C_type inactivation and closing rates was observed in the mutant T449K which, like the T449E channel, shows fast C-type inactivation (Lopez-Barneo et al.
We have studied in detail the dependence of closing on external K in the T449K channel, whose functional properties, like those of the mammalian homologous RCK4 which has a lysine at the position equivalent to 449, are strongly regulated by external [K] (Pardo et al.
In cells transfected with the T449K cDNA, high external K produced an increase of the macroscopic current amplitude and slowing of the inactivation time course (Fig.
The fast C_type inactivation of the T449K channel contrasts with its slow deactivation kinetics.
Effects of external K on permeability and closing kinetics of channel T449K A, representative traces of inward and outward tail currents recorded using the pulse protocol indicated above.
These findings indicate that whereas in the T449K channel external K can accede to the site of the C-inactivation gate and compete with inactivation, the cations cannot alter the activation-- deactivation gate.
This `anomalous' behaviour of the T449K channel, compared with what is seen in other pore mutants or some native K channels, could be the result of a nonuniform occupation of the channels by K, thus having different effects on the C_inactivation and closing gates located in different sites along the channel pore.
Figure 4 shows that the T449K channel retains a high K selectivity, with reversal potentials for K (-71 03 mV (n = 4) with 70 m external K) similar to the wild-type channel (see above).
2) the instantaneous I--V curves of the T449K channel are markedly non-linear, with saturation at negative membrane potentials even in the presence of 140 m external K.
The T449K channel can mediate large outward flow of K, but it has low conductivity in the inward direction.
Note that the activation rising phase is greatly prolonged in the wild-type channel (T449T) when compared with the progressively faster time course of channels T449K and D447E.
B, voltage dependence of the activation kinetics in T449T (0), T449K (7), T449E (6) and D447E () channel types.
7B) and T449K (Fig.
In agreement with these ideas, TEA has no effect on either inactivation or activation time courses in the mutant T449K (Fig.
1993) or in mutants T449K or T449E; however, it produces a clear deceleration of activation kinetics in mutant D447E.
For example, values for t of activation at +20 mV in the control (27 m K) solution were 201 035 ms (n = 9), 160 018 ms (n = 8), 139 014 ms (n = 9) and 061 015 ms (n = 8) for wild-type and channels T449K, T449E and D447E, respectively.
Interestingly, there are pore mutants (particularly the channel T449K) that C-inactivate at fast rates but close with DISCUSSION a slower time course than the wild-type channel.
Given that the T449K channels can mediate a large outward flow of K but have a low conductivity in the inward direction, it appears that the mutation leads to a non-uniform occupation of the channel by K: saturation of the sites accessible from the internal solution but low degree of occupancy of the sites accessible from the external solution.
These observations in the Sh mutant T449K are in excellent accord with the work of Ludewig et al.
1997), the low occupancy by cations of the external entrance to the pore in mutant T449K may be related in part to the Figure 7.
Effect of TEA on the activation and C-type inactivation time courses of three channel variants Recordings from the wild-type channel (A, T449T) can be compared with recordings from the D447E (B) and T449K (C) mutants.
Scaled current traces in the central panels were obtained at +20 mV with pulses of 11 s for T449T, 20 ms for D447E and 50 ms for T449K.
These same phenomena may favour the flow of ions in the outward direction which is a characteristic of the T449K channel.
D447E
protein
substitution
P08510
true positive
Mutant D447E had closing and C-type inactivation kinetics which were faster than the wildtype channel.
In this last position we studied RESULTS only the mutation that conserves the negative charge (D447E), since neutralization or reversion of the charge of this residue prevents the expression of K currents.
To study the effects of pore mutations on deactivation we first characterized the single mutant D447E whose C-type inactivation rate is about 500-fold faster than the wild-type channel (D447D) and is slowed by external K and TEA (see Molina et al.
The closing kinetics of the two 447 channel variants (D447D and D447E) are compared in Fig.
For example, at -70 mV, the deactivation time constant (c, see eqn (1)) of the D447E channel (048 009 ms, n = 6) was about 5 times faster than in the wild-type channel (206 065 ms, n = 8).
Single amino acid mutation with parallel changes in closing and C-type inactivation A, C-type inactivation time course of wild-type (D447D, left panel) and mutated (D447E, right panel) channels.
B, tail currents recorded in D447D (left) and D447E (right) channels with 30 m external K following a depolarizing pulse to +20 mV and repolarization to -90, -70 and -50 mV.
of eighteen (D447D, 1) and nine (D447E, 0) experiments.
(1) Since in both D447D and D447E channels macroscopic inactivation at membrane potentials more positive than 0 mV is voltage independent, we estimated the value of h from the inverse of the inactivation time constant measured at +20 mV.
The fact that in the mutant D447E both C-type inactivation and closing are faster than in the wild-type channel may indicate that the mutation produces a uniform decrease in K occupancy along the channel pore (see Swenson & Armstrong, 1981; Lopez-Barneo et al.
Effects of external K on permeability and closing kinetics of channel variants at position 447 A, representative traces of inward and outward tail currents recorded in channels D447D (left) and D447E (right) using the pulse protocol indicated in Fig.
B, instantaneous I--V relationship for 30 (0), 70 (6) and 140 (1) m external K in D447D (left) and D447E (right) channels.
The dependence of membrane conductance on K was more apparent in the mutant D447E, although in the range of membrane potentials studied and with high external K the rates of ion flow in the inward and outward directions were similar.
The reversal potentials of the K currents were not significantly different in the two channel types (-62 21 mV (n = 4) for D447D and -72 24 mV, (n = 4) for D447E with 70 m external K).
Thus, pore mutations such as D447E lead to parallel acceleration of the rates of C-inactivation and closing without affecting the selectivity of K over the other cations (mainly Na) in the recording solutions.
In the channel types D447D and D447E, closing and C-type inactivation rates were also affected in parallel by external K.
Pore mutations with differential effects on closing and C-type inactivation In contrast with the parallel effects of the mutation D447E on closing and C-type inactivation rates of Sh channels, we have found pore mutations which influence differentially these two kinetic processes.
509.2 Channel 1 type at 30 m K at 70 m K at 140 m K (ms) (ms) (ms) T449T (D447D) 26 08 (8) 41 06 (5) T449E 27 05 (5) 32 04 (3) T449K 40 07 (6) 42 06 (4) 43 (1) D447E 07 016 (6) 09 027 (4) 14 (1) was calculated from eqn (1).
Note that the activation rising phase is greatly prolonged in the wild-type channel (T449T) when compared with the progressively faster time course of channels T449K and D447E.
B, voltage dependence of the activation kinetics in T449T (0), T449K (7), T449E (6) and D447E () channel types.
7A) and in mutants D447E (Fig.
By contrast, in the mutant D447E, with a C-type inactivation rate hundreds of times faster than that for the wild-type channel, the slowing of C-type inactivation by TEA (Fig.
1993) or in mutants T449K or T449E; however, it produces a clear deceleration of activation kinetics in mutant D447E.
For example, values for t of activation at +20 mV in the control (27 m K) solution were 201 035 ms (n = 9), 160 018 ms (n = 8), 139 014 ms (n = 9) and 061 015 ms (n = 8) for wild-type and channels T449K, T449E and D447E, respectively.
In channels where C-type inactivation is very fast (as in mutant D447E), the net result of TEA or high external K is to slow down inactivation, which secondarily leads to deceleration of activation.
Effect of TEA on the activation and C-type inactivation time courses of three channel variants Recordings from the wild-type channel (A, T449T) can be compared with recordings from the D447E (B) and T449K (C) mutants.
Scaled current traces in the central panels were obtained at +20 mV with pulses of 11 s for T449T, 20 ms for D447E and 50 ms for T449K.
T449E
protein
substitution
P08510
true positive
For instance, the mutant T449E, Figure 3.
509.2 Channel 1 type at 30 m K at 70 m K at 140 m K (ms) (ms) (ms) T449T (D447D) 26 08 (8) 41 06 (5) T449E 27 05 (5) 32 04 (3) T449K 40 07 (6) 42 06 (4) 43 (1) D447E 07 016 (6) 09 027 (4) 14 (1) was calculated from eqn (1).
An even more marked separation between the changes of C_type inactivation and closing rates was observed in the mutant T449K which, like the T449E channel, shows fast C-type inactivation (Lopez-Barneo et al.
B, voltage dependence of the activation kinetics in T449T (0), T449K (7), T449E (6) and D447E () channel types.
1993) or in mutants T449K or T449E; however, it produces a clear deceleration of activation kinetics in mutant D447E.
For example, values for t of activation at +20 mV in the control (27 m K) solution were 201 035 ms (n = 9), 160 018 ms (n = 8), 139 014 ms (n = 9) and 061 015 ms (n = 8) for wild-type and channels T449K, T449E and D447E, respectively.
12773542
full text
H53Y
protein
substitution
true negative
In five cases (H53A, H53F, H53Y, Y124A, and H154Y), the maximal water permeability in cells expressing mutant AQP3 was significantly decreased compared with that in cells expressing wild-type AQP3.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
H129A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
S152A
protein
substitution
true negative
The Pf in cells expressing AQP3(W128A), AQP3(S152A), or AQP3(H241A) was similar to that in cells expressing wild-type AQP3.
In cells expressing AQP3(S152A), the range of pH sensitivity was shifted to more acidic pH (Fig.
At this pH, the Pf in cells expressing AQP3(S152A) was 3.9-fold higher compared with that in the surrounding untransfected cells.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
c and d, in contrast to cells expressing wild-type AQP3 (WT), the Pf in cells expressing AQP3(W128A), AQP3(H241A), or AQP3(S152A) was not decreased by 1 mM NiCl2.
d, the water permeability in cells expressing AQP3(S152A) was less responsive to acidic pH.
Y124A
protein
substitution
true negative
In five cases (H53A, H53F, H53Y, Y124A, and H154Y), the maximal water permeability in cells expressing mutant AQP3 was significantly decreased compared with that in cells expressing wild-type AQP3.
In cells expressing AQP3(H53A), AQP3(Y124A), or AQP3(H154F), the range of pH sensitivity was shifted to more alkaline values (Fig.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
Cells expressing AQP3(Y124A) had significantly lower maximal water permeability than cells expressing wild-type AQP3.
c, the mutation Y124A decreased the maximal water permeability of AQP3 and shifted the pH sensitivity curve to higher pH values.
H154Y
protein
substitution
true negative
In five cases (H53A, H53F, H53Y, Y124A, and H154Y), the maximal water permeability in cells expressing mutant AQP3 was significantly decreased compared with that in cells expressing wild-type AQP3.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
Y150A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
H53F
protein
substitution
true negative
In five cases (H53A, H53F, H53Y, Y124A, and H154Y), the maximal water permeability in cells expressing mutant AQP3 was significantly decreased compared with that in cells expressing wild-type AQP3.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
We found that AQP3(H154F), but not AQP3(H53F), had the same maximal water permeability as wild-type AQP3.
a, the Pf in cells expressing AQP3(H53A) or AQP3(H53F) was significantly lower than that in cells expressing wild-type AQP3 (WT).
H241A
protein
substitution
true negative
The Pf in cells expressing AQP3(W128A), AQP3(S152A), or AQP3(H241A) was similar to that in cells expressing wild-type AQP3.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
c and d, in contrast to cells expressing wild-type AQP3 (WT), the Pf in cells expressing AQP3(W128A), AQP3(H241A), or AQP3(S152A) was not decreased by 1 mM NiCl2.
D125A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
S233A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
D219A
protein
substitution
true negative
All mutants except D219A and W231A (Fig.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
AQP3(D219A) had a similar distribution.
W231A
protein
substitution
true negative
All mutants except D219A and W231A (Fig.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
a and b, AQP3(W231A) was retained in the endoplasmic reticulum of the cells.
W128A
protein
substitution
true negative
The Pf in cells expressing AQP3(W128A), AQP3(S152A), or AQP3(H241A) was similar to that in cells expressing wild-type AQP3.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
c and d, in contrast to cells expressing wild-type AQP3 (WT), the Pf in cells expressing AQP3(W128A), AQP3(H241A), or AQP3(S152A) was not decreased by 1 mM NiCl2.
D156A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
H154A
protein
substitution
true negative
In two cases (S49A and H154A), mutant AQP3 was water-impermeable.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
b, the mutation H154A rendered human AQP3 water-impermeable.
H154F
protein
substitution
true negative
In cells expressing AQP3(H53A), AQP3(Y124A), or AQP3(H154F), the range of pH sensitivity was shifted to more alkaline values (Fig.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
We found that AQP3(H154F), but not AQP3(H53F), had the same maximal water permeability as wild-type AQP3.
The maximal Pf in cells expressing AQP3(H154F) was similar to that in cells expressing wild-type AQP3, but was achieved at higher pH values.
H53A
protein
substitution
true negative
In five cases (H53A, H53F, H53Y, Y124A, and H154Y), the maximal water permeability in cells expressing mutant AQP3 was significantly decreased compared with that in cells expressing wild-type AQP3.
In cells expressing AQP3(H53A), AQP3(Y124A), or AQP3(H154F), the range of pH sensitivity was shifted to more alkaline values (Fig.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
a, the Pf in cells expressing AQP3(H53A) or AQP3(H53F) was significantly lower than that in cells expressing wild-type AQP3 (WT).
The range of pH sensitivity was shifted to higher pH values in cells expressing AQP3(H53A).
S138A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
D132A
protein
substitution
true negative
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
S49A
protein
substitution
true negative
In two cases (S49A and H154A), mutant AQP3 was water-impermeable.
Mutants Maximal Pf Change in pH sensitivity range s a nd pH 30041 Ni2 ensitivity Loop I S49A H53A H53F H53Y Loop II Y124A D125A W128A H129A D132A S138A Y150A S152A H154A H154F H154Y D156A Loop III D219A W231A S233A H241A Double mutations H53A/H154A H53F/H154F H53Y/H154Y H53F/H154Y H53Y/H154F a b 2a 2 2 2 2 2a NMb NMb 2a 2a 2a 2a 2a a 3 3 4 a 3 3 a a a a a a a NMb NM NM 2c NM 2c NMb NM NMb NMb NM 2c NMb NMb NMb NMb NMb The Pf was not different from the Pf in untransfected cells.
11598222
full text
T256R
protein
substitution
true positive
Q39128
A previous study has shown that two-point mutations in the pore region of KAT1 (T256R and G262K) function as dominant negative KAT1 mutations in X.
RESULTS Overexpression of Dominant Negative KAT1 Mutant Inhibits Light-Induced Stomatal Opening To investigate the suggested role of K in channels in stomatal opening in vivo, we initially generated transgenic Arabidopsis plants that express the kat1-T256R mutant under control of a single cauliflower mosaic virus (CaMV) 35S promoter.
Northern-blot analyses confirmed the expression of the kat1-T256R transgene (data not shown).
Because the single 35S promoter mediates moderate gene expression levels in Arabidopsis guard cells (Ichida et al., 1997; Allen et al., 1999), we subsequently used a plant expression vector containing a tandem repeat of the CaMV 35S promoter to increase the level of expression of the dominant negative KAT1 mutant, kat1-T256R.
Of these l i n e s , t w o t r a n s g e n i c K in d e p r e s s o r l i n e s ( k a t 1 T256R line nos.
We also chose another homozygous transgenic line (kat1-T256R no.
In contrast, stomatal apertures increased in the kat1-T256R line number 15-4 in the light by an average of only 29% and in line number 22-6 by an average of 35%.
However, amplification of KAT1 in transgenic lines was stronger, most likely due to expression of dominant negative kat1-T256R mutant transcripts in guard cells.
U Reduced K ptake in Transgenic Guard Cells To determine whether the dominant negative kat1T256R mutants reduce K uptake during lightinduced stomatal opening, epidermal strips were stained with sodium hexanitrocobaltate (III) (SHC), which is a K -specific stain that produces K granules in guard cells (Green et al., 1990).
MATERIALS AND METHODS Plant Transformation A dominant negative KAT1 mutant that has a point mutation at amino acid 256 (T256R; Baizabal-Aguirre et al., 1999) was subcloned into plant expression vectors (pBINJIT) containing a tandem repeat of CaMV 35S promoter (gift from Dr.
Two vectors and the two resulting constructs, pKAT1-T256R, either in the pBIN-JIT or in the pMON530 were introduced Plant Physiol.
G262K
protein
substitution
true positive
Q39128
A previous study has shown that two-point mutations in the pore region of KAT1 (T256R and G262K) function as dominant negative KAT1 mutations in X.
10556945
full text
R485Y
protein
substitution
true negative
5 On channels mutated on the residue R485 (R485Y) which is located on the external entryway of the pore the rupatadine-induced block did not decrease at potentials positive to +10 mV.
Wild-type hKv1.5 channels (WT) at the equivalent position (485) present arginine, and thus, rstly we studied whether the mutation R485Y aected rupatadine-induced block.
Voltage-dependence of activation of R485Y mutant channels was similar to that of WT channels, the Vh and the k values averaging 716.5+2.8 mV and 4.8+0.4 mV (n=5), respectively.
As it is shown in Figure 8b, in the presence of rupatadine R485Y channel activation became biphasic and similarly to that observed in WT channels, it shifted the Vh of the steeper component (k=4.0+0.2 mV) to more negative potentials (727.8+4.4 mV, n=5, P50.01), while the Vh of the shallow component averaged 111.4+10.7 mV.
The results indicated that at voltages at which the activation curve reached saturation in the absence of drug (40 mV) the blockade of R485Y channels was voltageindependent.
C a b al le r o e t a l Rupa ta dine on hKv1.5 c ha nne ls Figure 8 (a) Eects of 1 mM rupatadine on R485Y channels.
(b) Eects of 1 mM rupatadine on the voltagedependence of R485Y channels activation.
(c) Fractional block (f=IRUP/ICON) induced by 1 mM rupatadine on R485Y channels plotted as function of the membrane potential.
In an attempt to localize the possible residue involved in this external binding site we studied the eects of rupatadine on R485Y channels.
However, the total amount of block was not signicantly modied and the activation curve of R485Y channels in the presence of rupatadine still exhibited two components.
V512M
protein
substitution
true positive
P22460
In contrast, on V512M channels rupatadine reproduced all the features of the blockade observed on wild type channels.
We also studied the eects of rupatadine on V512M channels since it has been demonstrated that this residue is involved in the internal binding site of bupivacaine, a tertiary amine local anaesthetic (Franqueza et al., 1997).
Rupatadineinduced block of V512M channels at +40 mV averaged 40.9+6.0% (n=4) and similarly to what was observed on WT channels was voltage-dependent.
(d) Eects of 1 mM rupatadine on V512M channels.
(c) Current voltage relationship (500 ms isochronal) of V512M channels in the absence and in the presence of 1 mM rupatadine.
(f) Fractional block (f=IRUP/ICON) from data shown in panel e induced by 1 mM rupatadine on V512M channels plotted as function of the membrane potential.
In order to test this hypothesis, we performed experiments on V512M channels, a residue that is critical in the stereoselective binding of bupivacaine on hKv1.5 channels (Franqueza et al., 1997).
V512M mutation shifted &20 mV the voltage-dependence of channel activation in the negative direction and rupatadine-induced block also exhibited a marked voltage-dependent unblock.
12409506
full text
9351973
full text
11971066
full text
T666M
protein
substitution
true positive
O00555
Necrotising myositis was diagnosed based on muscle biopsy and cor ticosteroid treatment Japanese cases of familial hemiplegic migraine with cerebellar ataxia carrying a T666M mutation in the CACNA1A gene Familial hemiplegic migraine (FHM) is an autosomal dominantly inherited disorder characterised by migraine attacks preceded by transient hemiparesis.
A CT transition (T666M) in the CACNA1A gene was identified in both case 1 and case 2.
Recurrence of the T666M calcium channel CACNA1A gene mutation in familial hemiplegic migraine with progressive cerebellar ataxia.
Genetic features Mutational analyses of the CACNA1A gene were performed in cases 1 and 2 by direct nucleotide sequence analysis of exons 4, 16, 17, and 36, in which the first four missense mutations--namely, R192Q, T666M, V714A and I1811L--were repor ted.6 The analysis was www.jnnp.com 678 J Neurol Neurosurg Psychiatry 2002;72:675680 (MPNST).
V714A
protein
substitution
true positive
O00555
Genetic features Mutational analyses of the CACNA1A gene were performed in cases 1 and 2 by direct nucleotide sequence analysis of exons 4, 16, 17, and 36, in which the first four missense mutations--namely, R192Q, T666M, V714A and I1811L--were repor ted.6 The analysis was www.jnnp.com 678 J Neurol Neurosurg Psychiatry 2002;72:675680 (MPNST).
I1811L
protein
substitution
true negative
Genetic features Mutational analyses of the CACNA1A gene were performed in cases 1 and 2 by direct nucleotide sequence analysis of exons 4, 16, 17, and 36, in which the first four missense mutations--namely, R192Q, T666M, V714A and I1811L--were repor ted.6 The analysis was www.jnnp.com 678 J Neurol Neurosurg Psychiatry 2002;72:675680 (MPNST).
Sequence different from O00555
R192Q
protein
substitution
true positive
O00555
Genetic features Mutational analyses of the CACNA1A gene were performed in cases 1 and 2 by direct nucleotide sequence analysis of exons 4, 16, 17, and 36, in which the first four missense mutations--namely, R192Q, T666M, V714A and I1811L--were repor ted.6 The analysis was www.jnnp.com 678 J Neurol Neurosurg Psychiatry 2002;72:675680 (MPNST).
10899465
full text
C764R
protein
substitution
true negative
E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II).
To identify possible additional mutations, we amplified para cDNA fragments encoding the four transmem- Table 2 Knockdown times and para mutations among different German cockroach strains Strain para mutation L993Fa CSMA Orlando Ectiban-R Swine Aves Malo Pinellis 214 Pinellis 417 Fuerte NASJAX a b c d e f Time to knockdown (min) G330A G G G S/R A/A A/A E434K E E E S/R /E /E E/K E/K E/K E/K E/K C764R C C C S/R /C /C C/R C/R C/R C/R C/R P1880L P P P S/R P/P P/L P/P First fiveb 20c 18c 4570c 1520 1723 5081 837 1129 1448 1530 60 80 275 120 140 280 100 Last fiveb D58G D D D S/R f D/D D/G D/D L L F S/Rd L/Le /F L/F L/F L/F L/F /F Data taken from Dong et al.
Two additional amino acid changes, E434K and C764R, were found in all five of these strains.
The fact that the two amino acid changes (E434K and C764R) were detected only in the resistant individuals, but not in any susceptible individuals of any tested strain, strongly implicates 100% linkage of the two new mutations with the L993F mutation and their involvement in the knockdown resistance.
The E434K and C764R mutations are located in the first linker con- necting domains I and II.
E434K is situated close to the beginning of the linker region, whereas C764R is near the end of the linker (Fig.
Four additional mutations (D58G, E433K, C764R and P1880L, identified in this study) are associated with high levels of resistance.
L993F
protein
substitution
true negative
The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain.
Of the eight recently field-collected pyrethroidresistant German cockroach strains, five (Malo, Pinellis 214, Marietta, Swine and Fuerte) had been used in previous studies and were known to possess the L993F mutation (Dong et al., 1998), and three (Aves, Pinellis 417 and NASJAX) were collected from the field and characterized more recently (Valles, 1998; Valles et al., 2000).
Our previous study (Dong et al., 1998) showed that the kdr individuals in all of these strains possessed the L993F mutation.
To identify possible additional mutations, we amplified para cDNA fragments encoding the four transmem- Table 2 Knockdown times and para mutations among different German cockroach strains Strain para mutation L993Fa CSMA Orlando Ectiban-R Swine Aves Malo Pinellis 214 Pinellis 417 Fuerte NASJAX a b c d e f Time to knockdown (min) G330A G G G S/R A/A A/A E434K E E E S/R /E /E E/K E/K E/K E/K E/K C764R C C C S/R /C /C C/R C/R C/R C/R C/R P1880L P P P S/R P/P P/L P/P First fiveb 20c 18c 4570c 1520 1723 5081 837 1129 1448 1530 60 80 275 120 140 280 100 Last fiveb D58G D D D S/R f D/D D/G D/D L L F S/Rd L/Le /F L/F L/F L/F L/F /F Data taken from Dong et al.
As reported previously, our method was capable of detecting the L993F mutation if it was present in at least one of five pooled PCR reactions from five individuals (Dong et al., 1998).
We first confirmed the previous finding that the last five individuals of all five strains, Malo, Pinellis 214, Pinellis 417, Fuerte and NASJAX, had the L993F mutation.
The fact that the two amino acid changes (E434K and C764R) were detected only in the resistant individuals, but not in any susceptible individuals of any tested strain, strongly implicates 100% linkage of the two new mutations with the L993F mutation and their involvement in the knockdown resistance.
So far, the four additional kdr-associated mutations appear to coexist with the L993F mutation and have been Fig.
L993F was previously identified in a kdr strain (Ectiban-R) (Dong, 1997).
We sequenced para cDNA from a strain (Swine) that lacks the L993F mutation and exhibits only a modest level of kdr (Table 2).
These mutations also were not found in a kdr strain (Aves) that contained the L993F mutation (Valles et al., 2000) and exhibited only a moderate level of resistance (Table 2).
These mutations, together with the pre-existing L993F mutation, may be responsible for the high level of kdr resistance to pyrethroids in the German cockroach.
P1880L
protein
substitution
true negative
To identify possible additional mutations, we amplified para cDNA fragments encoding the four transmem- Table 2 Knockdown times and para mutations among different German cockroach strains Strain para mutation L993Fa CSMA Orlando Ectiban-R Swine Aves Malo Pinellis 214 Pinellis 417 Fuerte NASJAX a b c d e f Time to knockdown (min) G330A G G G S/R A/A A/A E434K E E E S/R /E /E E/K E/K E/K E/K E/K C764R C C C S/R /C /C C/R C/R C/R C/R C/R P1880L P P P S/R P/P P/L P/P First fiveb 20c 18c 4570c 1520 1723 5081 837 1129 1448 1530 60 80 275 120 140 280 100 Last fiveb D58G D D D S/R f D/D D/G D/D L L F S/Rd L/Le /F L/F L/F L/F L/F /F Data taken from Dong et al.
Compared with ParaCSMA, we found two additional amino acid changes, D58G and P1880L, in the Malo strain.
It is possible that the D58G and P1880L mutations detected in Malo are associated with the very high level of kdr in this strain.
The D58G and P1880L mutations are located at the nitrogen and carbon termini, respectively.
Four additional mutations (D58G, E433K, C764R and P1880L, identified in this study) are associated with high levels of resistance.
E433K
protein
substitution
true negative
Four additional mutations (D58G, E433K, C764R and P1880L, identified in this study) are associated with high levels of resistance.
E434K
protein
substitution
true negative
E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II).
To identify possible additional mutations, we amplified para cDNA fragments encoding the four transmem- Table 2 Knockdown times and para mutations among different German cockroach strains Strain para mutation L993Fa CSMA Orlando Ectiban-R Swine Aves Malo Pinellis 214 Pinellis 417 Fuerte NASJAX a b c d e f Time to knockdown (min) G330A G G G S/R A/A A/A E434K E E E S/R /E /E E/K E/K E/K E/K E/K C764R C C C S/R /C /C C/R C/R C/R C/R C/R P1880L P P P S/R P/P P/L P/P First fiveb 20c 18c 4570c 1520 1723 5081 837 1129 1448 1530 60 80 275 120 140 280 100 Last fiveb D58G D D D S/R f D/D D/G D/D L L F S/Rd L/Le /F L/F L/F L/F L/F /F Data taken from Dong et al.
Two additional amino acid changes, E434K and C764R, were found in all five of these strains.
The fact that the two amino acid changes (E434K and C764R) were detected only in the resistant individuals, but not in any susceptible individuals of any tested strain, strongly implicates 100% linkage of the two new mutations with the L993F mutation and their involvement in the knockdown resistance.
The E434K and C764R mutations are located in the first linker con- necting domains I and II.
E434K is situated close to the beginning of the linker region, whereas C764R is near the end of the linker (Fig.
D58G
protein
substitution
true negative
To identify possible additional mutations, we amplified para cDNA fragments encoding the four transmem- Table 2 Knockdown times and para mutations among different German cockroach strains Strain para mutation L993Fa CSMA Orlando Ectiban-R Swine Aves Malo Pinellis 214 Pinellis 417 Fuerte NASJAX a b c d e f Time to knockdown (min) G330A G G G S/R A/A A/A E434K E E E S/R /E /E E/K E/K E/K E/K E/K C764R C C C S/R /C /C C/R C/R C/R C/R C/R P1880L P P P S/R P/P P/L P/P First fiveb 20c 18c 4570c 1520 1723 5081 837 1129 1448 1530 60 80 275 120 140 280 100 Last fiveb D58G D D D S/R f D/D D/G D/D L L F S/Rd L/Le /F L/F L/F L/F L/F /F Data taken from Dong et al.
Compared with ParaCSMA, we found two additional amino acid changes, D58G and P1880L, in the Malo strain.
It is possible that the D58G and P1880L mutations detected in Malo are associated with the very high level of kdr in this strain.
The D58G and P1880L mutations are located at the nitrogen and carbon termini, respectively.
Furthermore, the D58G mutation is located at the nitrogen terminus.
Four additional mutations (D58G, E433K, C764R and P1880L, identified in this study) are associated with high levels of resistance.
G330A
protein
substitution
true negative
To identify possible additional mutations, we amplified para cDNA fragments encoding the four transmem- Table 2 Knockdown times and para mutations among different German cockroach strains Strain para mutation L993Fa CSMA Orlando Ectiban-R Swine Aves Malo Pinellis 214 Pinellis 417 Fuerte NASJAX a b c d e f Time to knockdown (min) G330A G G G S/R A/A A/A E434K E E E S/R /E /E E/K E/K E/K E/K E/K C764R C C C S/R /C /C C/R C/R C/R C/R C/R P1880L P P P S/R P/P P/L P/P First fiveb 20c 18c 4570c 1520 1723 5081 837 1129 1448 1530 60 80 275 120 140 280 100 Last fiveb D58G D D D S/R f D/D D/G D/D L L F S/Rd L/Le /F L/F L/F L/F L/F /F Data taken from Dong et al.
An additional amino acid change, G330A, was detected in strains Malo and Fuerte, but not in Pinellis 214, Pinellis 417 or NASJAX.
11159436
full text
V121T
protein
substitution
true positive
P35560-2
FIGURE 1 Sequence alignment in the pore (P) and PM regions of the inward rectifiers ROMK2, IRK1, C7, and C9 and the ROMK2 point mutants F129C, Q133E, A135P, V121T, and L117I.
It is not surprising that the point mutation V121T alters the sensitivity of ROMK to external K because this mutation was previously found to enhance the sensitivity of ROMK to external Ba and Cs (Zhou et al., 1996), increase single-channel conductance, and alter ion selectivity (Choe et al.
V121T and L117I are two point mutations constituting the difference between the ROMK and IRK pore (P) regions.
The initial conductances in 1 mM K were as follows: ROMK2, 102 33 S; V121T, 164 35 S; L117I, 106 21 S; C7, 140 15 S.
In the mutants F129C, E132D, Q133E, L117I, and V121T the effect of external cations is reduced.
The V121T mutation was previously shown to increase single-channel conductance and Ba block, but the L117I mutation did not affect these parameters.
A135P
protein
substitution
true positive
P35560-2
FIGURE 1 Sequence alignment in the pore (P) and PM regions of the inward rectifiers ROMK2, IRK1, C7, and C9 and the ROMK2 point mutants F129C, Q133E, A135P, V121T, and L117I.
The results of one of these mutations, ROMK2-A135P, is shown in Fig.
A135P (E) resembles wild-type ROMK2 (F) except that an Ala has been replaced by a Pro.
The initial conductances in 1 mM K were as follows: ROMK2, 102 33 S; A135P, 75 15 S; Q133E, 72 10 S.
Q133E
protein
substitution
true positive
P35560-2
FIGURE 1 Sequence alignment in the pore (P) and PM regions of the inward rectifiers ROMK2, IRK1, C7, and C9 and the ROMK2 point mutants F129C, Q133E, A135P, V121T, and L117I.
The point mutation E132D produced an effect similar to Q133E.
Q133E resembles wild-type ROMK2 except that a Gln at position 133 has been replaced by a negatively charged Glu.
The initial conductances in 1 mM K were as follows: ROMK2, 102 33 S; A135P, 75 15 S; Q133E, 72 10 S.
In the mutants F129C, E132D, Q133E, L117I, and V121T the effect of external cations is reduced.
K61M
protein
substitution
true positive
P35560-2
One clue to the action of Cs is that at FIGURE 16 A mutation at the cytoplasmic pH gate (K61M) blocks slow activation.
The ROMK2 mutation K61M is known to abolish the pH sensitivity of ROMK.
Initial conductances in 1 mM K solutions were as follows: ROMK2, 102 33 S; C9, 144 23 S; K61M, 207 57 S.
The ROMK2-K61M mutant cannot be protonated and is locked into the activated state.
Complete activation of outward conductance seems to require an intact pH sensor, because the K61M mutant of ROMK2 lacks both a pH gate and a slow increase in conductance (Fig.
Protonation of the cytoplasmic K61 site drives channels into the inactivated mode, unless this site has been altered (e.g., the mutation K61M).
E132D
protein
substitution
true positive
P35560-2
The point mutation E132D produced an effect similar to Q133E.
In the mutants F129C, E132D, Q133E, L117I, and V121T the effect of external cations is reduced.
L117I
protein
substitution
true positive
P35560-2
FIGURE 1 Sequence alignment in the pore (P) and PM regions of the inward rectifiers ROMK2, IRK1, C7, and C9 and the ROMK2 point mutants F129C, Q133E, A135P, V121T, and L117I.
The additional finding that the L117I mutation also eliminated the slow increase in outward conductance was unexpected as the L117I mutant did not have an increased affinity for external Ba (Zhou et al., 1996) or an altered single-channel conductance (Choe et al., 2000).
V121T and L117I are two point mutations constituting the difference between the ROMK and IRK pore (P) regions.
The initial conductances in 1 mM K were as follows: ROMK2, 102 33 S; V121T, 164 35 S; L117I, 106 21 S; C7, 140 15 S.
In the mutants F129C, E132D, Q133E, L117I, and V121T the effect of external cations is reduced.
The V121T mutation was previously shown to increase single-channel conductance and Ba block, but the L117I mutation did not affect these parameters.
F129C
protein
substitution
true positive
P35560-2
FIGURE 1 Sequence alignment in the pore (P) and PM regions of the inward rectifiers ROMK2, IRK1, C7, and C9 and the ROMK2 point mutants F129C, Q133E, A135P, V121T, and L117I.
The time course of F129C outward conducBiophysical Journal 80(2) 683 697 tance was similar to that of C9, except that the initial increase in conductance was slightly larger.
The initial conductances in 1 mM K were as follows: ROMK2, 102 33 S; F129C, 113 22 S; C9, 144 23 S.
12) and the ROMK2-F129C mutant (Fig.
In the mutants F129C, E132D, Q133E, L117I, and V121T the effect of external cations is reduced.
11159437
full text
Y401F
protein
substitution
true positive
P15390
Based upon the G and the fact that Y401F does not change neoSTX affinity (Favre et al., 1995), the Tyr-401/N1-OH interaction seems likely to result from a "hydrogen" bond between the N1-OH and the e lectrons of the aromatic ring.
E755A
protein
substitution
true positive
P15390
Some of the STX affinity determinations for I and all determinations for D400A, E755A, and D1532N were previously reported by Penzotti et al.
Because of their low toxin affinities, kinetic measurements with E755A and D1532N were felt to be affected by the bath exchange rate and were not further analyzed.
Y401C caused a TABLE 1 Comparison of the effects of channel mutations on neoSTX and STX blocking efficacy IC50 SEM (nM) 0.1 1.1 6.2 25.3 989.7 0.1 2263.6 0.5 155.0 13.7 12.5 5277.7 3.0 13078.5 n 9 8 8 8 4 10 4 13 10 10 5 8 12 8 IC50 Ratio 1 41 267 659 20499 4 88939 1 469 41 77 13423 11 31094 kon 8.4 4.0 2.4 5.2 5.5 SEM (nM 10 10 10 10 10 10 10 10 10 10 3 4 5 5 1 Channel Mutation neoSTX I D400A Y401D Y401C E755A M1240A D1532N STX I D400A Y401D Y401C E755A M1240A D1532N s 1) 10 10 10 10 10 10 10 10 10 10 4 4 6 5 n 7 4 7 6 10 3.8 8.2 5.2 1.4 1.0 koff 10 10 10 10 10 10 10 10 10 10 SEM (s 1) 3 3 3 2 n 4 3 3 3 Kd SEM (nM) 0.1 6.3 30.7 64.4 0.3 1.8 1461.5 73.9 70.6 5.6 n 7 4 7 6 10 0.4 16.3 106.8 263.4 8199.5 1.7 35575.7 4.1 1924.9 169.3 314.4 55035.8 44.7 127487.2 8.5 1.0 4.4 1.0 1.2 1.5 3.3 6.2 1.4 1.6 2.3 1.1 1.0 2.5 8.1 7.4 1.7 5.1 3.4 1.6 10 10 10 10 10 10 10 10 10 10 8 4 7 6 10 0.5 24.3 231.9 306.9 2.4 3 3 2 4 4.3 1.8 2.4 6.5 5.9 3 6 5 5 3 7 6 5 8 6 6 4 10 1.3 1.0 7.1 2.2 1.3 2 2 3 2 4 3 4 3 9 6 6 4 10 6.3 6524.8 373.6 376.7 29.9 8 6 6 4 10 4 4 2 3 Biophysical Journal 80(2) 698 706 NeoSTX Interactions with the Sodium Channel 701 FIGURE 2 Comparison of IC50 values of STX (black bars) and neoSTX (striped bars) for the native I and seven outer vestibule mutations showing the relative effect of adding the N1-OH group.
Y401D
protein
substitution
true positive
P15390
A similar change was seen with Y401D.
Y401C caused a TABLE 1 Comparison of the effects of channel mutations on neoSTX and STX blocking efficacy IC50 SEM (nM) 0.1 1.1 6.2 25.3 989.7 0.1 2263.6 0.5 155.0 13.7 12.5 5277.7 3.0 13078.5 n 9 8 8 8 4 10 4 13 10 10 5 8 12 8 IC50 Ratio 1 41 267 659 20499 4 88939 1 469 41 77 13423 11 31094 kon 8.4 4.0 2.4 5.2 5.5 SEM (nM 10 10 10 10 10 10 10 10 10 10 3 4 5 5 1 Channel Mutation neoSTX I D400A Y401D Y401C E755A M1240A D1532N STX I D400A Y401D Y401C E755A M1240A D1532N s 1) 10 10 10 10 10 10 10 10 10 10 4 4 6 5 n 7 4 7 6 10 3.8 8.2 5.2 1.4 1.0 koff 10 10 10 10 10 10 10 10 10 10 SEM (s 1) 3 3 3 2 n 4 3 3 3 Kd SEM (nM) 0.1 6.3 30.7 64.4 0.3 1.8 1461.5 73.9 70.6 5.6 n 7 4 7 6 10 0.4 16.3 106.8 263.4 8199.5 1.7 35575.7 4.1 1924.9 169.3 314.4 55035.8 44.7 127487.2 8.5 1.0 4.4 1.0 1.2 1.5 3.3 6.2 1.4 1.6 2.3 1.1 1.0 2.5 8.1 7.4 1.7 5.1 3.4 1.6 10 10 10 10 10 10 10 10 10 10 8 4 7 6 10 0.5 24.3 231.9 306.9 2.4 3 3 2 4 4.3 1.8 2.4 6.5 5.9 3 6 5 5 3 7 6 5 8 6 6 4 10 1.3 1.0 7.1 2.2 1.3 2 2 3 2 4 3 4 3 9 6 6 4 10 6.3 6524.8 373.6 376.7 29.9 8 6 6 4 10 4 4 2 3 Biophysical Journal 80(2) 698 706 NeoSTX Interactions with the Sodium Channel 701 FIGURE 2 Comparison of IC50 values of STX (black bars) and neoSTX (striped bars) for the native I and seven outer vestibule mutations showing the relative effect of adding the N1-OH group.
The coupling energy, G, was 1.3 0.1 kcal/mol ( 0.1), and a similar G was seen with Y401D.
Moreover, a similar small coupling between the N1-OH site and Y401D was observed ( 0.4).
Y401C
protein
substitution
true positive
P15390
(1995) reported an apparent inhibition constant of 275 nM for Y401C, similar to our result of 263 25 nM.
The channel mutation, Y401C, caused a 77-fold increase in the STX IC50 to 314 13 nM.
If Y401C and the N1-OH group of neoSTX were distant from each other, then the effects of substitutions on the IC50 values should have been independent.
Y401C caused a TABLE 1 Comparison of the effects of channel mutations on neoSTX and STX blocking efficacy IC50 SEM (nM) 0.1 1.1 6.2 25.3 989.7 0.1 2263.6 0.5 155.0 13.7 12.5 5277.7 3.0 13078.5 n 9 8 8 8 4 10 4 13 10 10 5 8 12 8 IC50 Ratio 1 41 267 659 20499 4 88939 1 469 41 77 13423 11 31094 kon 8.4 4.0 2.4 5.2 5.5 SEM (nM 10 10 10 10 10 10 10 10 10 10 3 4 5 5 1 Channel Mutation neoSTX I D400A Y401D Y401C E755A M1240A D1532N STX I D400A Y401D Y401C E755A M1240A D1532N s 1) 10 10 10 10 10 10 10 10 10 10 4 4 6 5 n 7 4 7 6 10 3.8 8.2 5.2 1.4 1.0 koff 10 10 10 10 10 10 10 10 10 10 SEM (s 1) 3 3 3 2 n 4 3 3 3 Kd SEM (nM) 0.1 6.3 30.7 64.4 0.3 1.8 1461.5 73.9 70.6 5.6 n 7 4 7 6 10 0.4 16.3 106.8 263.4 8199.5 1.7 35575.7 4.1 1924.9 169.3 314.4 55035.8 44.7 127487.2 8.5 1.0 4.4 1.0 1.2 1.5 3.3 6.2 1.4 1.6 2.3 1.1 1.0 2.5 8.1 7.4 1.7 5.1 3.4 1.6 10 10 10 10 10 10 10 10 10 10 8 4 7 6 10 0.5 24.3 231.9 306.9 2.4 3 3 2 4 4.3 1.8 2.4 6.5 5.9 3 6 5 5 3 7 6 5 8 6 6 4 10 1.3 1.0 7.1 2.2 1.3 2 2 3 2 4 3 4 3 9 6 6 4 10 6.3 6524.8 373.6 376.7 29.9 8 6 6 4 10 4 4 2 3 Biophysical Journal 80(2) 698 706 NeoSTX Interactions with the Sodium Channel 701 FIGURE 2 Comparison of IC50 values of STX (black bars) and neoSTX (striped bars) for the native I and seven outer vestibule mutations showing the relative effect of adding the N1-OH group.
The effect of eliminating the negative charge of Asp-400 by mutagenesis was dependent upon the presence of the N1-OH group, but the changes in kon caused by Y401C were similar in STX or neoSTX.
The channel mutation, Y401C, has a much larger effect on STX blocking efficacy than it does on neoSTX.
The observed equivalent decrease in the toxin kon seen with Y401C for both STX and neoSTX must be the result of effects other than that between the N1-OH group and Tyr-401.
(A) Current records from an oocyte injected with Y401C.
The mean kon values are shown for STX and neoSTX for Y401C, native I, and D400A.
D400A
protein
substitution
true positive
P15390
Some of the STX affinity determinations for I and all determinations for D400A, E755A, and D1532N were previously reported by Penzotti et al.
Y401C caused a TABLE 1 Comparison of the effects of channel mutations on neoSTX and STX blocking efficacy IC50 SEM (nM) 0.1 1.1 6.2 25.3 989.7 0.1 2263.6 0.5 155.0 13.7 12.5 5277.7 3.0 13078.5 n 9 8 8 8 4 10 4 13 10 10 5 8 12 8 IC50 Ratio 1 41 267 659 20499 4 88939 1 469 41 77 13423 11 31094 kon 8.4 4.0 2.4 5.2 5.5 SEM (nM 10 10 10 10 10 10 10 10 10 10 3 4 5 5 1 Channel Mutation neoSTX I D400A Y401D Y401C E755A M1240A D1532N STX I D400A Y401D Y401C E755A M1240A D1532N s 1) 10 10 10 10 10 10 10 10 10 10 4 4 6 5 n 7 4 7 6 10 3.8 8.2 5.2 1.4 1.0 koff 10 10 10 10 10 10 10 10 10 10 SEM (s 1) 3 3 3 2 n 4 3 3 3 Kd SEM (nM) 0.1 6.3 30.7 64.4 0.3 1.8 1461.5 73.9 70.6 5.6 n 7 4 7 6 10 0.4 16.3 106.8 263.4 8199.5 1.7 35575.7 4.1 1924.9 169.3 314.4 55035.8 44.7 127487.2 8.5 1.0 4.4 1.0 1.2 1.5 3.3 6.2 1.4 1.6 2.3 1.1 1.0 2.5 8.1 7.4 1.7 5.1 3.4 1.6 10 10 10 10 10 10 10 10 10 10 8 4 7 6 10 0.5 24.3 231.9 306.9 2.4 3 3 2 4 4.3 1.8 2.4 6.5 5.9 3 6 5 5 3 7 6 5 8 6 6 4 10 1.3 1.0 7.1 2.2 1.3 2 2 3 2 4 3 4 3 9 6 6 4 10 6.3 6524.8 373.6 376.7 29.9 8 6 6 4 10 4 4 2 3 Biophysical Journal 80(2) 698 706 NeoSTX Interactions with the Sodium Channel 701 FIGURE 2 Comparison of IC50 values of STX (black bars) and neoSTX (striped bars) for the native I and seven outer vestibule mutations showing the relative effect of adding the N1-OH group.
With D400A, the kon values were 4.0 10 4 nM 1 s 1 for both pH 7.2 (n 4) and pH 6.0 (n 2), respectively.
At pH 7.2, D400A resulted in only a twofold increase in the neoSTX koff rate, and this effect was not altered by changes in pH.
The D400A koff values were 8.2 10 3 s 1 (n 4) and 3 1 5.1 10 s (n 2) at pH 7.2 and 6.0, respectively.
The mean kon values are shown for STX and neoSTX for Y401C, native I, and D400A.
D1532N
protein
substitution
true positive
P15390
Some of the STX affinity determinations for I and all determinations for D400A, E755A, and D1532N were previously reported by Penzotti et al.
Because of their low toxin affinities, kinetic measurements with E755A and D1532N were felt to be affected by the bath exchange rate and were not further analyzed.
Y401C caused a TABLE 1 Comparison of the effects of channel mutations on neoSTX and STX blocking efficacy IC50 SEM (nM) 0.1 1.1 6.2 25.3 989.7 0.1 2263.6 0.5 155.0 13.7 12.5 5277.7 3.0 13078.5 n 9 8 8 8 4 10 4 13 10 10 5 8 12 8 IC50 Ratio 1 41 267 659 20499 4 88939 1 469 41 77 13423 11 31094 kon 8.4 4.0 2.4 5.2 5.5 SEM (nM 10 10 10 10 10 10 10 10 10 10 3 4 5 5 1 Channel Mutation neoSTX I D400A Y401D Y401C E755A M1240A D1532N STX I D400A Y401D Y401C E755A M1240A D1532N s 1) 10 10 10 10 10 10 10 10 10 10 4 4 6 5 n 7 4 7 6 10 3.8 8.2 5.2 1.4 1.0 koff 10 10 10 10 10 10 10 10 10 10 SEM (s 1) 3 3 3 2 n 4 3 3 3 Kd SEM (nM) 0.1 6.3 30.7 64.4 0.3 1.8 1461.5 73.9 70.6 5.6 n 7 4 7 6 10 0.4 16.3 106.8 263.4 8199.5 1.7 35575.7 4.1 1924.9 169.3 314.4 55035.8 44.7 127487.2 8.5 1.0 4.4 1.0 1.2 1.5 3.3 6.2 1.4 1.6 2.3 1.1 1.0 2.5 8.1 7.4 1.7 5.1 3.4 1.6 10 10 10 10 10 10 10 10 10 10 8 4 7 6 10 0.5 24.3 231.9 306.9 2.4 3 3 2 4 4.3 1.8 2.4 6.5 5.9 3 6 5 5 3 7 6 5 8 6 6 4 10 1.3 1.0 7.1 2.2 1.3 2 2 3 2 4 3 4 3 9 6 6 4 10 6.3 6524.8 373.6 376.7 29.9 8 6 6 4 10 4 4 2 3 Biophysical Journal 80(2) 698 706 NeoSTX Interactions with the Sodium Channel 701 FIGURE 2 Comparison of IC50 values of STX (black bars) and neoSTX (striped bars) for the native I and seven outer vestibule mutations showing the relative effect of adding the N1-OH group.
The addition of the hydroxyl group to position 1 of the 1,2,3 guanidinium group had more modest effects on the change in affinity seen with domain III Met-1240 and domain IV D1532N ( G 0.5 0.1 kcal/mol and G 0.7 0.1 kcal/mol, respectively).
The large effect of D1532N on STX binding compared with that of TTX (Penzotti et al., 1998) and the modest coupling of the 1-OH, 2,3 guanidinium site and Asp-1532 ( G 0 7 0.1 kcal/mol), consistent with the modest change in the charge distribution increasing the positive charge on the hydrogen at the N2 site with addition of the N1-OH, provided experimental evidence to support the suggestion that the 1,2,3 guanidinium group is directed toward domain IV.
E403Q
protein
substitution
true positive
P15390
Limitations on the supply of neoSTX precluded accurate determination of the IC50 values for E403Q and E758Q.
M1240A
protein
substitution
true positive
P15390
Y401C caused a TABLE 1 Comparison of the effects of channel mutations on neoSTX and STX blocking efficacy IC50 SEM (nM) 0.1 1.1 6.2 25.3 989.7 0.1 2263.6 0.5 155.0 13.7 12.5 5277.7 3.0 13078.5 n 9 8 8 8 4 10 4 13 10 10 5 8 12 8 IC50 Ratio 1 41 267 659 20499 4 88939 1 469 41 77 13423 11 31094 kon 8.4 4.0 2.4 5.2 5.5 SEM (nM 10 10 10 10 10 10 10 10 10 10 3 4 5 5 1 Channel Mutation neoSTX I D400A Y401D Y401C E755A M1240A D1532N STX I D400A Y401D Y401C E755A M1240A D1532N s 1) 10 10 10 10 10 10 10 10 10 10 4 4 6 5 n 7 4 7 6 10 3.8 8.2 5.2 1.4 1.0 koff 10 10 10 10 10 10 10 10 10 10 SEM (s 1) 3 3 3 2 n 4 3 3 3 Kd SEM (nM) 0.1 6.3 30.7 64.4 0.3 1.8 1461.5 73.9 70.6 5.6 n 7 4 7 6 10 0.4 16.3 106.8 263.4 8199.5 1.7 35575.7 4.1 1924.9 169.3 314.4 55035.8 44.7 127487.2 8.5 1.0 4.4 1.0 1.2 1.5 3.3 6.2 1.4 1.6 2.3 1.1 1.0 2.5 8.1 7.4 1.7 5.1 3.4 1.6 10 10 10 10 10 10 10 10 10 10 8 4 7 6 10 0.5 24.3 231.9 306.9 2.4 3 3 2 4 4.3 1.8 2.4 6.5 5.9 3 6 5 5 3 7 6 5 8 6 6 4 10 1.3 1.0 7.1 2.2 1.3 2 2 3 2 4 3 4 3 9 6 6 4 10 6.3 6524.8 373.6 376.7 29.9 8 6 6 4 10 4 4 2 3 Biophysical Journal 80(2) 698 706 NeoSTX Interactions with the Sodium Channel 701 FIGURE 2 Comparison of IC50 values of STX (black bars) and neoSTX (striped bars) for the native I and seven outer vestibule mutations showing the relative effect of adding the N1-OH group.
The small change in STX blocking efficacy seen as a result of M1240A was of similar magnitude to those seen with other neutral mutations of Met-1240 (Perez-Garca et al., i 1996; Terlau et al., 1991), suggesting that the N1-OH site of STX is probably not interacting strongly with Met-1240 under native conditions.
E758Q
protein
substitution
true positive
P15390
Limitations on the supply of neoSTX precluded accurate determination of the IC50 values for E403Q and E758Q.
10366610
full text
I1495F
protein
substitution
true positive
P35499
Introduction of the I1495F mutation into the wild-type channels disrupted the macroscopic current inactivation decay and shifted both steady-state activation and inactivation to the hyperpolarizing direction.
Additionally, a significant enhancement of slow inactivation was observed in the I1495F mutation.
These results, showing that the I1495F and T704M hyperkalaemic periodic paralysis mutations both have profound effects on channel activation and fastslow inactivation, suggest that the S5 segment maybe in a location where fast and slow inactivation converge.
Stabl y ex pressi ng c el l li nes were produc ed usi ng the ex pression vector pRc-C M V encodi ng either w ild-t y pe (W T) human skeletal muscle voltage-gated sodium channel or the cor respondi ng I1495F mutant construct (Bendah hou et al., 1995).
Constr uction of hSkM1-I1495F and hSkM1-T704M.
T he hSkM1-I1495F was constructed usi ng the megapr i mer PCR method of site-di rected mutagenesi s (Sarkar and Sommer, 1990).
T he pr i mer contai ned a nucleotide substitution T4561C (i ndicated above by an italic letter), which results i n the ami no acid substitution I1495F.
A 1.3 kb product was i solated from a 1% agarose gel usi ng the Qiagen (Hilden, Germany) gel ex traction k it, cut w ith SseI restr iction enz y me and ligated to the appropr iatel y cut fragment of w ild-t y pe hSkM1 (the pRc-C M V-hSkM1 construct was i nitial l y cut w ith SseI enz y me to separate a 10 kb band from the 1.3 kb w ild-t y pe band) to construct the f ul l-leng th mutated channel hSkM1-I1495F.
To check for the presence of thi s component in the hSkM1-I1495F channels, cells were first held at 120 mV and then depolari z ed to different potentials for 25 msec.
2 A) and the hSkM1-I1495F (Fig.
Effect of I1495F mutation on macroscopic currents.
C ells were held at 120 mV, and inward sodium currents were elicited by 10 mV voltage steps from 80 to 20 mV for W T ( A) and I1495F ( B).
C shows normali z ed current traces from W T and I1495F mutant recorded at 10 mV.
D, A n example of superimposed current recordings from W T and I1495F channels using the same pulse protocol as in AC for a 100 msec duration.
Inacti vation kinetics of I1495F.
A, Time constant of fast inacti vation for W T ( filled circles, n 39) and I1495F (open circles, n 14) show ing an additional slow component (open squares, n 14) for the H y perK PP mutant channels.
H EK 293 cells were subjected to a protocol described in Materials and Methods to generate the inacti vation cur ves for W T ( filled circles, n 43) and I1495F (open circles, n 31).
A single-ex ponential f unction was sufficient for fitting w ild-t y pe currents, whereas a t wo-ex ponential f unction was necessar y to obtain an accurate fit of sustained I1495F currents (Fig.
T he midpoint of 62.1 4.9 mV; inacti vation was shifted by 10 mV (W T, V0.5 n 43; I1495F, V0.5 72.3 4.3 mV; n 31), and thi s significant shift ( p 0.001) was not transient.
However, there i s no significant change ( p 0.1) in 3he slope of the inacti vation cur ve for I1495F channels (W T, z t .9 0.7; n 26; I1495F, z 3.7 0.5; n 30) as reported for the different substitutions at position 1448 of the human skeletal muscle sodium channel.
(at 10 mV) from w ild-t y pe and I1495F channels are superimposed to show the defect in the fast inacti vation decay.
Figure 4 A shows the currentvoltage relationship for W T and I1495F channels.
T he I1495F channels acti vate at more negati ve potentials.
T he steady-state acti vation i s significantly shifted ( p 0 1 .001) to the left for the hSkM1-I1495F by 8 mV (W T, V0.5 25.5 3.7 mV; n 31).
7.7 3.0 mV; n 47; I1495F, V0.5 Fig ure 4.
W T and I1495F acti vation parameters.
A, T he currentvoltage cur ves for W T ( filled circles, n 47) and I1495F (open circles, n 31) were normali z ed for a better appreciation of the shift bet ween the t wo relations.
Deacti vation, development, and recover y from inacti vation for I1495F.
Resulting currents were fitted by a single-ex ponential decay and ex pressed as f unction of the voltage for W T ( filled circles, n 20) and for I1495F (open circles, n 20).
Peak currents obtained using the test pulse were normali z ed to the peak current obtained during the inacti vating pulse for W T ( filled circles, n 14) and I1495F (open circles, n 26).
T he averaged time constants from W T ( filled s ymbols, n 10) and I1495F (open s ymbols, n 7) are show n.
I1495F enhances slow inacti vation.
A, Steady-state slow inacti vation in hSkM1 W T ( filled circles, n 8) and I1495F (open circles, n 8 ) a fter 50 sec conditioning pulse ranging from 130 to 10 mV.
B and C show development of slow inacti vation and recover y from slow inacti vation, respecti vely, as a f unction of time for W T ( filled circles, n 7) and I1495F (open circles, n 7) channels.
There was no significant change ( p 0.05) in the apparent gating charge bet ween the t wo constructs (W T, z 4.2 0.6; n 47; I1495F, z 4.0 0.4; n 31).
However, when testing for recover y from fast inacti vation, the fraction of channels recovered a fter a prolonged 80 mV conditioning pulse was smaller for I1495F channels than for the W T channels (Fig.
However, development of fast inacti vation seems to be enhanced at negati ve voltages in the I1495F channels, indicating that closed-state inacti vation i s enhanced in the I1495F mutation.
I1495F enhances slow inactivation It has been proposed that a di sruption of slow inacti vation i s also necessar y to account for the ex tended duration of paralysi s seen in patients w ith H y perK PP (Ruff, 1994).
Results from I1495F slow inacti vation ex periments are show n in Figure 6.
Our data show that steady-state slow inacti vation (s ) i s not impai red for I1495F channels as reported for the rat T698M mutation (C ummins and Sig worth, 1996), but in contrast, i s Deactivation, development, and recover y from fast inactivation in I1495F A defect in channel deacti vation may favor prolonged action potentials, as show n in a model of muscle membrane excitabilit y (Featherstone et al., 1998).
A s show n in Figure 5, kinetics of deacti vation of the I1495F channels are not different from W T Bendahhou et al.
enhanced w ith the I1495F mutant (W T, V0.5 61.6 2.8 mV; z 0 2.7 0.2; n 8; I1495F, V0.5 76.7 2.6 mV; z 1.8 .1; n 8) (Fig.
6C) do not seem to be significantly altered by the I1495F mutation.
Hence, we were interested in the compari son of the T704M and I1495F mutations in the human background and in the same ex pression system.
T he shift in acti vation i s similar to what has been reported prev iously for T704M channels (Yang et al., 1994) and for rT698M channels (C ummins et al., 1993), and to the shift in acti vation caused by the I1495F mutation.
We also compared tail current kinetics of W T and T704M under the same conditions as those used for the I1495F.
8 A, B), in contrast to the I1495F mutant in which deacti vation was not a ffected.
Our data on the recover y from fast inacti vation show that the T704M recovers from fast inacti vation in a manner similar to the I1495F mutant.
8C), whereas the I1495F ex hibits a faster development of closed-state inacti vation.
Because the rat T698M and the I1495F mutations have altered slow inacti vation and because the T704M i s located at the intracellular inter face of the S5 segment of domain II, we tested whether slow inacti vation of human skeletal muscle sodium channels was a ffected by the T704M mutation.
E x pression of the I1495F mutation in H EK 293 cells revealed alterations in channel f unction that could account for the onset of H y perK PP in our patient.
C omparing I1495F and T704M mutations revealed a prominent role of the S5 segment in channel acti vation, as well as in fast and slow inacti vation.
Macroscopic inward sodium current from cells ex pressing I1495F showed that thi s substitution results in a sustained current at a w ide range of physiological voltages.
T he slowly decay ing component measured in I1495F suggests that the S5 segment of domain I V i s involved in channel inacti vation.
T hi s behav ior i s surpri sing because I1495F i s embedded in the membrane, presumably far from the intracellular membrane inter face and also far from the III-I V linker, a strong candidate for the fast inacti vation gate of the sodium channel (Stuhmer et al., 1989; West et al., 1992).
A lthough initially we do obser ve larger sustained currents in I1495F channels, these currents appear to run dow n, and a fter 10 min in the whole-cell configuration, we do not find any difference in the si z e of W T and I1495F sustained currents.
Inacti vation, especially at negati ve potentials, i s significantly enhanced in the I1495F construct but not a ffected in T704M; thi s i s indicated by a 10 mV hy perpolari z ing shift in the steady-state fast inacti vation of I1495F compared w ith W T and the faster development of inacti vation at negati ve voltages ( 80 to 60 mV, Fig.
T he protocols used for the T704M are the same as those described for the I1495F mutation in Figure 5.
T hese data show that the T704M mutation di srupts slow inacti vation in human SkM1 channels in an identical manner to the effect of the rT698M mutation on rat SkM1 channels but di stinct from the I1495F mutation in which slow inacti vation was particularly enhanced.
T he acti vation cur ve was not changed by the M1360V mutation, in contrast to the I1495F and T704M mutations that shifted the voltage dependence of acti vation toward negati ve potentials.
T he similarit y obser ved here bet ween the I1495F and T704M mutations in the acti vation cur ve may suggest a role of the S5 segments (at least in domain II and I V) in channel acti vation.
However, although myotonia i s prominent in patients w ith the T704M mutation, the patient w ith the I1495F mutation did not ex hibit myotonia.
Whereas the T704M mutation significantly ( p 0.001) slowed deacti vation, the I1495F mutation had much less of an effect compared w ith W T.
We investigated also the rate of recover y from fast inacti vation in W T, I1495F, and T704M.
Whereas the I1495F has a slower recover y rate than W T (most prominent at 80 mV), the T704M mutation did not significantly alter the rate of recover y from fast inacti vation.
We found that slow inacti vation was differentially di srupted in the I1495F and T704M mutants.
In the I1495F channels, in contrast, slow inacti vation i s dramatically enhanced; thi s i s show n by the left shift in the steady-state slow inacti vation.
Because the patient w ith the I1495F mutation did not ex hibit myotonia, our data on slow inacti vation suggests that di sruption of slow inacti vation could be crucial to the development of myotonia in H y perK PP patients.
In contrast to most patients w ith H y perK PP who t y pically have painless epi sodes of paralysi s, the patient w ith the I1495F mutation ex perienced crampy pain during the attack s of weak ness.
T herefore, we propose that intermittent acti vation of a ffected muscle fibers during attack s of weak ness might contribute to the crampy pain associated w ith the I1495F mutation.
Because the I1495F mutation enhances slow inacti vation, our results demonstrate that the manifestation of H y perK PP does not necessarily requi re di sruption of slow inacti vation.
R1448P
protein
substitution
true positive
P35499
T hi s hy perpolari z ing shift of the inacti vation cur ve has not been reported in a pure H y perK PP mutant but was rather seen in mutations occurring at position 1448, R1448P, C, H (C hahine et al., 1994; Yang et al., 1994), or S (S.
R1448S
protein
substitution
true positive
P35499
Defecti ve deacti vation may well account for sodium channel di sease, as reported in our ow n study on the R1448S mutation (Bendahhou, C ummins, Kw iecinski, Wa x man, and Ptcek, una publi shed obser vations) and studies from other laboratories (Richmond et al., 1997; Featherstone et al., 1998).
T4561C
protein
substitution
true negative
T he pr i mer contai ned a nucleotide substitution T4561C (i ndicated above by an italic letter), which results i n the ami no acid substitution I1495F.
Sequence analysi s of the aberrant conformer revealed a T4561C mutation (data not show n) of the SCN4A gene that predicts an i soleucineto-phenylalanine change at position 1495 in the human skeletal muscle sodium channel -subunit.
I693T
protein
substitution
true positive
P35499
Interestingly, another mutation in the S4 S5 linker of domain II, I693T, also shifts acti vation in the negati ve di rection and induces weak ness but apparently not myotonia (Plassart-Schiess et al., 1998).
M1360V
protein
substitution
true positive
P35499
Fi ve point mutations have been reported to a ffect the human sodium channel gene SCN4A causing H y perK PP: T704M (C annon and Strittmatter, 1993; C ummins et al., 1993; Yang et al., 1994; C ummins and Sig worth, 1996; Hay ward et al., 1997), V781I (Baquero et al., 1995), A1156T (Yang et al., 1994), M1360V (Wagner et al., 1997), and M1592V (Hay ward et al., 1997).
However, the M1360V mutation responsible for H y perK PP and PC (Wagner et al., 1997) causes a 13 mV shift in channel availabilit y only.
T he acti vation cur ve was not changed by the M1360V mutation, in contrast to the I1495F and T704M mutations that shifted the voltage dependence of acti vation toward negati ve potentials.
T698M
protein
substitution
true positive
P15390
Subsequently, it was demonstrated that the rT698M mutation does indeed impai r slow inacti vation of the rat SkM1 sodium channel (C ummins and Sig worth, 1996).
Our data show that steady-state slow inacti vation (s ) i s not impai red for I1495F channels as reported for the rat T698M mutation (C ummins and Sig worth, 1996), but in contrast, i s Deactivation, development, and recover y from fast inactivation in I1495F A defect in channel deacti vation may favor prolonged action potentials, as show n in a model of muscle membrane excitabilit y (Featherstone et al., 1998).
A lthough the corresponding mutation has been ex tensi vely studied in the rat clone (rT698M; C annon and Strittmatter, 1993; C ummins et al., 1993; C ummins and Sig worth, 1996; Hay ward et al., 1997), the T704M mutation in the human clone has not been f ully characteri z ed (Yang et al., 1994).
T he shift in acti vation i s similar to what has been reported prev iously for T704M channels (Yang et al., 1994) and for rT698M channels (C ummins et al., 1993), and to the shift in acti vation caused by the I1495F mutation.
We have show n prev iously that there i s no difference bet ween rat W T and rat T698M channels in terms of the voltage dependence of steady-state fast inacti vation (C ummins et al., 1993; C ummins and Sig worth, 1996).
Because the rat T698M and the I1495F mutations have altered slow inacti vation and because the T704M i s located at the intracellular inter face of the S5 segment of domain II, we tested whether slow inacti vation of human skeletal muscle sodium channels was a ffected by the T704M mutation.
T hese data show that the T704M mutation di srupts slow inacti vation in human SkM1 channels in an identical manner to the effect of the rT698M mutation on rat SkM1 channels but di stinct from the I1495F mutation in which slow inacti vation was particularly enhanced.
(1993) found a similar result for the rat T698M mutation (equi valent to the human T704M causing H y perK PP).
A1156T
protein
substitution
true positive
P35499
Fi ve point mutations have been reported to a ffect the human sodium channel gene SCN4A causing H y perK PP: T704M (C annon and Strittmatter, 1993; C ummins et al., 1993; Yang et al., 1994; C ummins and Sig worth, 1996; Hay ward et al., 1997), V781I (Baquero et al., 1995), A1156T (Yang et al., 1994), M1360V (Wagner et al., 1997), and M1592V (Hay ward et al., 1997).
M1592V
protein
substitution
true positive
P35499
Fi ve point mutations have been reported to a ffect the human sodium channel gene SCN4A causing H y perK PP: T704M (C annon and Strittmatter, 1993; C ummins et al., 1993; Yang et al., 1994; C ummins and Sig worth, 1996; Hay ward et al., 1997), V781I (Baquero et al., 1995), A1156T (Yang et al., 1994), M1360V (Wagner et al., 1997), and M1592V (Hay ward et al., 1997).
T704M
protein
substitution
true positive
P35499
Sig worth for supporting some of the work on the T704M mutation.
In contrast, the T704M mutation, a hyperkalaemic periodic paralysis mutation located in the cytoplasmic interface of the S5 segment of the second domain, also shifted activation in the hyperpolarizing direction but had little effect on fast inactivation and dramatically impaired slow inactivation.
These results, showing that the I1495F and T704M hyperkalaemic periodic paralysis mutations both have profound effects on channel activation and fastslow inactivation, suggest that the S5 segment maybe in a location where fast and slow inactivation converge.
Fi ve point mutations have been reported to a ffect the human sodium channel gene SCN4A causing H y perK PP: T704M (C annon and Strittmatter, 1993; C ummins et al., 1993; Yang et al., 1994; C ummins and Sig worth, 1996; Hay ward et al., 1997), V781I (Baquero et al., 1995), A1156T (Yang et al., 1994), M1360V (Wagner et al., 1997), and M1592V (Hay ward et al., 1997).
We have also per formed additional comparati ve ex periments on the most common H y perK PP mutation (T704M), located at the intracellular inter face on the S5 segment of the sodium channel.
Constr uction of hSkM1-I1495F and hSkM1-T704M.
T he hSkM1-T704M construct was engi neered as descr ibed prev iousl y (Yang et al., 1994) Electrophysiolog y.
Effect of the T704M mutation on hSk6M1 currents.
Family of traces from representati ve H EK 293 cells ex pressing either W T ( A) or T704M ( B) channels.
C, Peak currentvoltage relationship for W T ( filled squares, n 8) and T704M (open squares, n 7).
D, T he steady-state fast inactivation cur ves for W T ( filled circles, n 12) and T704M (open circles, n 10) channels w ith 500 msec inacti vating prepulses are show n.
C urrents were converted to conductance, from the currentvoltage cur ve show n in Figure 7C, for W T ( filled squares, n 8 ) and T704M (open squares, n 7), and fit was made according to the Bolt z mann f unction.
The T704M mutation T he T704M i s the most common H y perK PP mutation and i s located at the intracellular inter face on the S5 segment of domain II.
A lthough the corresponding mutation has been ex tensi vely studied in the rat clone (rT698M; C annon and Strittmatter, 1993; C ummins et al., 1993; C ummins and Sig worth, 1996; Hay ward et al., 1997), the T704M mutation in the human clone has not been f ully characteri z ed (Yang et al., 1994).
For example, it i s not k now n whether the T704M mutation alters deacti vation or slow inacti vation of human skeletal muscle sodium channels.
Hence, we were interested in the compari son of the T704M and I1495F mutations in the human background and in the same ex pression system.
7A) or the T704M channels (Fig.
T he voltage dependence of acti vation and conductance was significantly different ( p 0.005) bet ween W T (V0.5 19.0 1.7 mV; n 28) and T704M (V0.5 26.9 1.8 mV; n 26) in H EK 293 cells (Fig.
T he apparent gating charge was also significantly different ( p 0.005) bet ween W T (z 3.7 0.1; n 15) and T704M (z 3.0 0.2; n 13) channels.
T he shift in acti vation i s similar to what has been reported prev iously for T704M channels (Yang et al., 1994) and for rT698M channels (C ummins et al., 1993), and to the shift in acti vation caused by the I1495F mutation.
At 0 mV, where w indow currents are not ex pected to contribute to the sustained current, we did not find any difference bet ween the si z e of the sustained currents in W T (0.4 0.8% of peak; n 8) and T704M (0.3 0.7% of peak; n 1 0) cells.
In contrast, at 50 mV, where w indow currents can contribute to the persi stent current, the sustained currents were larger in T704M cells (0.5 0.2% of peak; n 10) than in W T c 0ells (0.1 0.2% of peak; n 8).
We found no difference ( p .3) bet ween steady-state fast inacti vation of W T ( 70.5 1.2; z 4.0 0.1; n 31) and T704M ( 73.6 1.1; z 3.6 0.1; n 29) channels (Fig.
We were caref ul to measure steadystate fast inacti vation at the same time point in the recording for both W T and T704M channels.
We also compared tail current kinetics of W T and T704M under the same conditions as those used for the I1495F.
We found that the time constant of deacti vation was slower in the T704M than in the W T channels (Fig.
T hi s suggests that the T704M mutation alters the transition bet ween the closed and open states.
Our data on the recover y from fast inacti vation show that the T704M recovers from fast inacti vation in a manner similar to the I1495F mutant.
However, development of fast closed-state inacti vation seems to be una ffected by the T704M mutation (Fig.
Because the rat T698M and the I1495F mutations have altered slow inacti vation and because the T704M i s located at the intracellular inter face of the S5 segment of domain II, we tested whether slow inacti vation of human skeletal muscle sodium channels was a ffected by the T704M mutation.
9A), 50% of the T704M current remains available for acti vation under the same conditions (Fig.
We obser ved a significant impai rment of slow inacti vation for T704M channels compared w ith W T channels.
C omparing I1495F and T704M mutations revealed a prominent role of the S5 segment in channel acti vation, as well as in fast and slow inacti vation.
We did not obser ve large sustained currents in T704M channels at 0 mV but only at more negati ve voltages at which w indow currents might occur.
Inacti vation, especially at negati ve potentials, i s significantly enhanced in the I1495F construct but not a ffected in T704M; thi s i s indicated by a 10 mV hy perpolari z ing shift in the steady-state fast inacti vation of I1495F compared w ith W T and the faster development of inacti vation at negati ve voltages ( 80 to 60 mV, Fig.
Deacti vation, development, and recover y from fast inacti vation for the T704M.
T he protocols used for the T704M are the same as those described for the I1495F mutation in Figure 5.
A, Tail current traces obtained from W T and T704M channels at 70 mV.
B, Time constants of tail current as show n in A were plotted as a f unction of the test potential for W T ( filled circles, n 5) and T704M (open circles, n 6).
C, Development of fast inacti vation (squares) and recover y from fast inactivation (circles) for W T ( filled s ymbols, n 8) and T704M (open s ymbols, n 7) were obtained as described in Figure 5.
In contrast, at 0 mV, 50 10% (n 7 ) of the T704M current i s available for acti vation a fter the 30 msec recover y pulse.
T he T704M channels recovered from slow inacti vation much faster than did the W T 4hannels (Fig.
With a recover y time of 100 msec, only 11 c 5% (n 5) of the W T current recovers, whereas 66 9% (n ) of T704M current recovers.
T he initial rate of recover y for the T704M channels i s similar to that for the recover y from fast inacti vation.
Figure 9E shows the time course for the development of slow inacti vation for W T and T704M channels.
At 20 mV, the time constant was significantly ( p 0.05) smaller for W T channels (3.9 0.7 sec; n 5) than for T704M channels (12.0 2.9 sec; n 5).
T hese data show that the T704M mutation di srupts slow inacti vation in human SkM1 channels in an identical manner to the effect of the rT698M mutation on rat SkM1 channels but di stinct from the I1495F mutation in which slow inacti vation was particularly enhanced.
Slow inacti vation i s impai red in T704M cells.
C hanges in peak sodium current for representati ve W T ( A) and T704M ( B) cells in response to changes in the holding potential.
C, T he steady-state slow inacti vation cur ves for W T ( filled circles, n 8) and T704M (open circles, n 7) cells are show n.
D, Normal5z ed Na current in representati ve W T ( filled circles, n i ) and T704M (open circles, n 6) cells recovering from slow inacti vation.
E, Development of slow inacti vation i s show n to be slower for the T704M (open circles, n 5) than for the W T channels ( filled circles, n 5).
T he acti vation cur ve was not changed by the M1360V mutation, in contrast to the I1495F and T704M mutations that shifted the voltage dependence of acti vation toward negati ve potentials.
(1993) found a similar result for the rat T698M mutation (equi valent to the human T704M causing H y perK PP).
T he similarit y obser ved here bet ween the I1495F and T704M mutations in the acti vation cur ve may suggest a role of the S5 segments (at least in domain II and I V) in channel acti vation.
However, although myotonia i s prominent in patients w ith the T704M mutation, the patient w ith the I1495F mutation did not ex hibit myotonia.
Whereas the T704M mutation significantly ( p 0.001) slowed deacti vation, the I1495F mutation had much less of an effect compared w ith W T.
We investigated also the rate of recover y from fast inacti vation in W T, I1495F, and T704M.
Whereas the I1495F has a slower recover y rate than W T (most prominent at 80 mV), the T704M mutation did not significantly alter the rate of recover y from fast inacti vation.
We found that slow inacti vation was differentially di srupted in the I1495F and T704M mutants.
Slow inacti vation appears to be incomplete in T704M channels; a fter prolonged depolari z ations lasting 20 min or more, more than one-thi rd of T704M channels fail to become slow inacti vated and are available for acti vation a fter a brief (e.g., 30 msec) hy perpolari z ation.
V781I
protein
substitution
true positive
P35499
Fi ve point mutations have been reported to a ffect the human sodium channel gene SCN4A causing H y perK PP: T704M (C annon and Strittmatter, 1993; C ummins et al., 1993; Yang et al., 1994; C ummins and Sig worth, 1996; Hay ward et al., 1997), V781I (Baquero et al., 1995), A1156T (Yang et al., 1994), M1360V (Wagner et al., 1997), and M1592V (Hay ward et al., 1997).
A recent study of the V781I mutation in human embr yonic kidney (H EK) 293 cells suggested that thi s mutation may be a benign poly morphi sm (Green et al., 1997).
9729613
full text
T338A
protein
substitution
true positive
P13569
Significant rectification resulting in a -I + 50I-50 ratio significantly less than one was observed in all mutants except T338A (Fig.
Single CFTR channel currents in inside-out patches excised from CHO cells stably expressing wild-type, T338A or T338S are shown in Fig.
Mean slope conductance was increased from 79 01 pS (n = 18) for wild-type to 104 01 pS (n = 9) for T338A and 113 02 pS (n = 5) for T338S.
Although the gating of T338A and T338S channels was not studied in detail, no striking differences from wild-type were noted.
Unitary properties of T338A and T338S CFTR A, examples of single channel activity for wild-type, T338A and T338S, recorded at -50 mV.
B and C, mean single channel current--voltage relationships for wild-type, T338A (B) and T338S (C).
Permeability of intracellular anions in wild-type and mutant CFTR Cl channels 7 Anion WT T338A T338S T338N T338V T339V Thiocyanate 263 013 (6) 585 027 (4) * 480 019 (3) * 872 103 (4) * 192 035 (4) * 328 008 (4) * Nitrate 153 004 (7) 204 008 (3) * 182 003 (4) * 422 022 (3) * 684 118 (7) * 161 002 (3) Bromide 123 003 (5) 174 004 (3) * 147 007 (3) * 166 015 (3) * 104 009 (5) 139 006 (4) * Chloride 100 001 (10) 100 002 (11) 100 002 (6) 100 003 (10) 100 004 (11) 100 006 (10) Iodide 084 003 (5) 209 016 (5) * 176 009 (3) * 103 005 (3) * 079 011 (3) 084 002 (3) Perchlorate 025 002 (6) 135 008 (3) * 066 006 (3) * 041 003 (3) * 054 000 (3) * 024 001 (4) Benzoate 0069 0006 (6) 017 003 (4) * 0091 0019 (3) 0089 0015 (4) 015 002 (4) * 0097 0014 (4) Hexafluorophosphate < 0019 (4) 053 001 (3) * 031 002 (3) * 068 002 (3) * 039 005 (3) * 0051 0010 (4) * Fluoride 011 001 (7) 012 002 (4) 0095 0012 (4) 011 001 (4) 0093 0009 (3) 017 002 (4) * Formate 025 001 (8) 045 004 (3) * 043 003 (3) * 035 004 (4) * 022 001 (3) 028 002 (3) Acetate 0090 0004 (8) 019 001 (3) * 018 001 (3) * 010 002 (5) 011 002 (3) 016 001 (3) * Propanoate 014 001 (3) 018 002 (4) 0098 0010 (4) * 0077 0013 (3) * 013 002 (3) Pyruvate 010 001 (5) 020 001 (3) * 013 002 (3) 0075 0015 (3) 017 003 (3) * Methane sulphonate 0077 0005 (5) 014 002 (4) * 0079 0014 (3) 0038 0004 (3) * 0088 0007 (3) Glutamate 0096 0008 (4) 0082 0009 (3) 0080 0008 (3) 0060 0012 (5) * 011 001 (3) Isethionate 013 001 (4) 011 001 (3) 0086 0012 (5) * 0043 0007 (3) * 0067 0005 (3) * Gluconate 0068 0004 (36) 010 001 (3) * 0060 0004 (3) 0044 0004 (3) 0077 0009 (3) 0088 0021 (5) Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.
Hanrahan Wild-type SCN > NO > Br > Cl > I > ClO > formate > F > PF T338A SCN > I NO > Br > ClO > Cl > PF > formate > F T338S SCN > NO I > Br > Cl > ClO > formate > PF > F T338N SCN > NO > Br > I = Cl > PF > ClO > formate > F T338V NO > SCN > Br = Cl > I > ClO > PF > formate > F J.
Note that all lyotropic anions showed the permeability sequence T338A > T338S > wild-type, again suggesting that the effects of these mutations on pore properties are correlated with the size of the amino acid side chain substituted.
In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0528 nm (wild-type), 0576 nm (T338A), 0549 nm (T338S), 0510 nm (T338N) and 0540 nm (T338V).
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0528 nm for wild-type, 0576 nm for T338A, 0549 nm for T338S, 0510 nm for T338N and 0540 nm for T338V.
In this case, fits by eqn (2) suggested minimum pore diameters of 0535 nm (wildtype), 0615 nm (T338A), 0505 nm (T338S), 0503 nm (T338N) and 0530 nm (T338V).
As with T338A and T338S, T339V showed apparently normal channel gating, with open probability being time and Figure 9.
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0535 nm for wild type, 0615 nm for T338A, 0505 nm for T338S, 0503 nm for T338N and 0530 nm for T338V.
Conversely, the elevated conductance of T338A and T338S might be advantageous in gene or protein replacement therapies for Figure 10.
Interestingly, both T338A (104 pS; Fig.
Thus in T338A, for example, the relationship between permeability and ionic hydration energy appeared much more linear than for wild-type (Fig.
Relationship between relative permeability and free energy of hydration for different intracellular anions A, wild-type; B, T338A.
T338F
protein
substitution
true positive
P13569
Stable expression of all six T338 mutants constructed in BHK cells led to the production of both coreglycosylated (band B) and fully glycosylated (band C) CFTR protein, although some mutants (especially T338F) J.
In contrast, no currents were activated under these conditions in large inside-out patches excised from BHK cells expressing T338F (n = 8; not shown).
Thus, the lack of macroscopic Cl currents in membrane patches excised from BHK cells expressing T338F (see above) may reflect a non-conducting phenotype for this mutant.
Alternatively, misprocessing or degradation of T338F protein (Fig.
T338N
protein
substitution
true positive
P13569
Example macroscopic current--voltage relationships for wild-type and T338 mutant CFTRs, recorded with symmetrical Cl-containing solutions Note the inward rectification observed with some T338 mutants, especially T338N.
Rectification was particularly strong in T338N (-I+ 50I-50 = 033 005; n = 6).
However, clear single channel currents were never resolved for T338N, T338V or T338I, either in CHO cell patches or in patches excised from BHK cells selected using a lower concentration of MTX (20 ), at potentials as hyperpolarized as -100 mV (data not shown).
We attempted to make some estimate of the conductance of T338N, T338V and T338I channels by analysing the increase in current noise associated with macroscopic current activation (Fig.
5C and D) and T338N and T338V (data not shown) Cl currents was associated with only a very small increase in noise.
5D) yielded chord conductances at -50 mV of 023 002 pS (n = 4) for T338N, 036 010 pS (n = 5) for T338V, and 017 004 pS (n = 5) for T338I.
Permeability of intracellular anions in wild-type and mutant CFTR Cl channels 7 Anion WT T338A T338S T338N T338V T339V Thiocyanate 263 013 (6) 585 027 (4) * 480 019 (3) * 872 103 (4) * 192 035 (4) * 328 008 (4) * Nitrate 153 004 (7) 204 008 (3) * 182 003 (4) * 422 022 (3) * 684 118 (7) * 161 002 (3) Bromide 123 003 (5) 174 004 (3) * 147 007 (3) * 166 015 (3) * 104 009 (5) 139 006 (4) * Chloride 100 001 (10) 100 002 (11) 100 002 (6) 100 003 (10) 100 004 (11) 100 006 (10) Iodide 084 003 (5) 209 016 (5) * 176 009 (3) * 103 005 (3) * 079 011 (3) 084 002 (3) Perchlorate 025 002 (6) 135 008 (3) * 066 006 (3) * 041 003 (3) * 054 000 (3) * 024 001 (4) Benzoate 0069 0006 (6) 017 003 (4) * 0091 0019 (3) 0089 0015 (4) 015 002 (4) * 0097 0014 (4) Hexafluorophosphate < 0019 (4) 053 001 (3) * 031 002 (3) * 068 002 (3) * 039 005 (3) * 0051 0010 (4) * Fluoride 011 001 (7) 012 002 (4) 0095 0012 (4) 011 001 (4) 0093 0009 (3) 017 002 (4) * Formate 025 001 (8) 045 004 (3) * 043 003 (3) * 035 004 (4) * 022 001 (3) 028 002 (3) Acetate 0090 0004 (8) 019 001 (3) * 018 001 (3) * 010 002 (5) 011 002 (3) 016 001 (3) * Propanoate 014 001 (3) 018 002 (4) 0098 0010 (4) * 0077 0013 (3) * 013 002 (3) Pyruvate 010 001 (5) 020 001 (3) * 013 002 (3) 0075 0015 (3) 017 003 (3) * Methane sulphonate 0077 0005 (5) 014 002 (4) * 0079 0014 (3) 0038 0004 (3) * 0088 0007 (3) Glutamate 0096 0008 (4) 0082 0009 (3) 0080 0008 (3) 0060 0012 (5) * 011 001 (3) Isethionate 013 001 (4) 011 001 (3) 0086 0012 (5) * 0043 0007 (3) * 0067 0005 (3) * Gluconate 0068 0004 (36) 010 001 (3) * 0060 0004 (3) 0044 0004 (3) 0077 0009 (3) 0088 0021 (5) Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.
Hanrahan Wild-type SCN > NO > Br > Cl > I > ClO > formate > F > PF T338A SCN > I NO > Br > ClO > Cl > PF > formate > F T338S SCN > NO I > Br > Cl > ClO > formate > PF > F T338N SCN > NO > Br > I = Cl > PF > ClO > formate > F T338V NO > SCN > Br = Cl > I > ClO > PF > formate > F J.
However, the permeabilities of the low conductance mutants T338N and T338V were more difficult to interpret, possibly indicating that substitution of a larger amino acid for T338 causes a more severe disruption of pore function.
In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0528 nm (wild-type), 0576 nm (T338A), 0549 nm (T338S), 0510 nm (T338N) and 0540 nm (T338V).
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0528 nm for wild-type, 0576 nm for T338A, 0549 nm for T338S, 0510 nm for T338N and 0540 nm for T338V.
In this case, fits by eqn (2) suggested minimum pore diameters of 0535 nm (wildtype), 0615 nm (T338A), 0505 nm (T338S), 0503 nm (T338N) and 0530 nm (T338V).
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0535 nm for wild type, 0615 nm for T338A, 0505 nm for T338S, 0503 nm for T338N and 0530 nm for T338V.
6), although the low conductances estimated for T338N, T338V and T338I should be considered rough approximations only.
All single channel conductances reported in this paper were measured at hyperpolarized potentials; conductance of the mutant channels T338N, T338V and T338I might be significantly lower at depolarized potentials (Fig.
T338I
protein
substitution
true positive
P13569
However, clear single channel currents were never resolved for T338N, T338V or T338I, either in CHO cell patches or in patches excised from BHK cells selected using a lower concentration of MTX (20 ), at potentials as hyperpolarized as -100 mV (data not shown).
We attempted to make some estimate of the conductance of T338N, T338V and T338I channels by analysing the increase in current noise associated with macroscopic current activation (Fig.
512.1 In contrast to wild-type, activation of macroscopic T338I (Fig.
5D) yielded chord conductances at -50 mV of 023 002 pS (n = 4) for T338N, 036 010 pS (n = 5) for T338V, and 017 004 pS (n = 5) for T338I.
T338I gave only very small currents when intracellular Cl was replaced by other anions, such that accurate measurement of Vrev was not possible, and therefore the selectivity of this mutant was not studied further.
Estimation of unitary current amplitude from macroscopic current noise A and C, activation of macroscopic CFTR current at -50 mV is associated with a large increase in current noise for wild-type (A), but a much smaller increase in noise for T338I (C).
6), although the low conductances estimated for T338N, T338V and T338I should be considered rough approximations only.
The low conductance of T338I could also explain why this mutation is associated with CF (Saba et al.
All single channel conductances reported in this paper were measured at hyperpolarized potentials; conductance of the mutant channels T338N, T338V and T338I might be significantly lower at depolarized potentials (Fig.
T339Y
protein
substitution
true positive
P13569
Similar results were observed with T339A, T339Y and T339F (not shown).
Indeed, we found no PKA- and ATP-dependent currents in very large inside-out patches excised from BHK cells expressing T339A (n = 6), T339S (n = 4), T339Y (n = 6) or T339F (n = 5) using symmetrical 150 m NaCl-containing solutions (not shown).
T339A
protein
substitution
true positive
P13569
Similar results were observed with T339A, T339Y and T339F (not shown).
Indeed, we found no PKA- and ATP-dependent currents in very large inside-out patches excised from BHK cells expressing T339A (n = 6), T339S (n = 4), T339Y (n = 6) or T339F (n = 5) using symmetrical 150 m NaCl-containing solutions (not shown).
Both T339A (McDonough et al.
1997b), raising the possibility that the detrimental effects of the T339A mutation on processing may be `rescued' by simultaneous mutation of T338.
K335E
protein
substitution
true positive
P13569
K335E (Tabcharani et al.
T339C
protein
substitution
true positive
P13569
1994) and T339C (Cheung & Akabas, 1996) mutant channels can be expressed in Xenopus oocytes following injection of in vitro transcribed cRNA.
T338S
protein
substitution
true positive
P13569
Single CFTR channel currents in inside-out patches excised from CHO cells stably expressing wild-type, T338A or T338S are shown in Fig.
Mean slope conductance was increased from 79 01 pS (n = 18) for wild-type to 104 01 pS (n = 9) for T338A and 113 02 pS (n = 5) for T338S.
Although the gating of T338A and T338S channels was not studied in detail, no striking differences from wild-type were noted.
Unitary properties of T338A and T338S CFTR A, examples of single channel activity for wild-type, T338A and T338S, recorded at -50 mV.
B and C, mean single channel current--voltage relationships for wild-type, T338A (B) and T338S (C).
Permeability of intracellular anions in wild-type and mutant CFTR Cl channels 7 Anion WT T338A T338S T338N T338V T339V Thiocyanate 263 013 (6) 585 027 (4) * 480 019 (3) * 872 103 (4) * 192 035 (4) * 328 008 (4) * Nitrate 153 004 (7) 204 008 (3) * 182 003 (4) * 422 022 (3) * 684 118 (7) * 161 002 (3) Bromide 123 003 (5) 174 004 (3) * 147 007 (3) * 166 015 (3) * 104 009 (5) 139 006 (4) * Chloride 100 001 (10) 100 002 (11) 100 002 (6) 100 003 (10) 100 004 (11) 100 006 (10) Iodide 084 003 (5) 209 016 (5) * 176 009 (3) * 103 005 (3) * 079 011 (3) 084 002 (3) Perchlorate 025 002 (6) 135 008 (3) * 066 006 (3) * 041 003 (3) * 054 000 (3) * 024 001 (4) Benzoate 0069 0006 (6) 017 003 (4) * 0091 0019 (3) 0089 0015 (4) 015 002 (4) * 0097 0014 (4) Hexafluorophosphate < 0019 (4) 053 001 (3) * 031 002 (3) * 068 002 (3) * 039 005 (3) * 0051 0010 (4) * Fluoride 011 001 (7) 012 002 (4) 0095 0012 (4) 011 001 (4) 0093 0009 (3) 017 002 (4) * Formate 025 001 (8) 045 004 (3) * 043 003 (3) * 035 004 (4) * 022 001 (3) 028 002 (3) Acetate 0090 0004 (8) 019 001 (3) * 018 001 (3) * 010 002 (5) 011 002 (3) 016 001 (3) * Propanoate 014 001 (3) 018 002 (4) 0098 0010 (4) * 0077 0013 (3) * 013 002 (3) Pyruvate 010 001 (5) 020 001 (3) * 013 002 (3) 0075 0015 (3) 017 003 (3) * Methane sulphonate 0077 0005 (5) 014 002 (4) * 0079 0014 (3) 0038 0004 (3) * 0088 0007 (3) Glutamate 0096 0008 (4) 0082 0009 (3) 0080 0008 (3) 0060 0012 (5) * 011 001 (3) Isethionate 013 001 (4) 011 001 (3) 0086 0012 (5) * 0043 0007 (3) * 0067 0005 (3) * Gluconate 0068 0004 (36) 010 001 (3) * 0060 0004 (3) 0044 0004 (3) 0077 0009 (3) 0088 0021 (5) Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.
Hanrahan Wild-type SCN > NO > Br > Cl > I > ClO > formate > F > PF T338A SCN > I NO > Br > ClO > Cl > PF > formate > F T338S SCN > NO I > Br > Cl > ClO > formate > PF > F T338N SCN > NO > Br > I = Cl > PF > ClO > formate > F T338V NO > SCN > Br = Cl > I > ClO > PF > formate > F J.
Note that all lyotropic anions showed the permeability sequence T338A > T338S > wild-type, again suggesting that the effects of these mutations on pore properties are correlated with the size of the amino acid side chain substituted.
In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0528 nm (wild-type), 0576 nm (T338A), 0549 nm (T338S), 0510 nm (T338N) and 0540 nm (T338V).
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0528 nm for wild-type, 0576 nm for T338A, 0549 nm for T338S, 0510 nm for T338N and 0540 nm for T338V.
In this case, fits by eqn (2) suggested minimum pore diameters of 0535 nm (wildtype), 0615 nm (T338A), 0505 nm (T338S), 0503 nm (T338N) and 0530 nm (T338V).
As with T338A and T338S, T339V showed apparently normal channel gating, with open probability being time and Figure 9.
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0535 nm for wild type, 0615 nm for T338A, 0505 nm for T338S, 0503 nm for T338N and 0530 nm for T338V.
Conversely, the elevated conductance of T338A and T338S might be advantageous in gene or protein replacement therapies for Figure 10.
4B) and T338S (113 pS; Fig.
Thus T338S has the highest conductance of any CFTR variant described to date.
T338V
protein
substitution
true positive
P13569
However, clear single channel currents were never resolved for T338N, T338V or T338I, either in CHO cell patches or in patches excised from BHK cells selected using a lower concentration of MTX (20 ), at potentials as hyperpolarized as -100 mV (data not shown).
We attempted to make some estimate of the conductance of T338N, T338V and T338I channels by analysing the increase in current noise associated with macroscopic current activation (Fig.
5C and D) and T338N and T338V (data not shown) Cl currents was associated with only a very small increase in noise.
5D) yielded chord conductances at -50 mV of 023 002 pS (n = 4) for T338N, 036 010 pS (n = 5) for T338V, and 017 004 pS (n = 5) for T338I.
Permeability of intracellular anions in wild-type and mutant CFTR Cl channels 7 Anion WT T338A T338S T338N T338V T339V Thiocyanate 263 013 (6) 585 027 (4) * 480 019 (3) * 872 103 (4) * 192 035 (4) * 328 008 (4) * Nitrate 153 004 (7) 204 008 (3) * 182 003 (4) * 422 022 (3) * 684 118 (7) * 161 002 (3) Bromide 123 003 (5) 174 004 (3) * 147 007 (3) * 166 015 (3) * 104 009 (5) 139 006 (4) * Chloride 100 001 (10) 100 002 (11) 100 002 (6) 100 003 (10) 100 004 (11) 100 006 (10) Iodide 084 003 (5) 209 016 (5) * 176 009 (3) * 103 005 (3) * 079 011 (3) 084 002 (3) Perchlorate 025 002 (6) 135 008 (3) * 066 006 (3) * 041 003 (3) * 054 000 (3) * 024 001 (4) Benzoate 0069 0006 (6) 017 003 (4) * 0091 0019 (3) 0089 0015 (4) 015 002 (4) * 0097 0014 (4) Hexafluorophosphate < 0019 (4) 053 001 (3) * 031 002 (3) * 068 002 (3) * 039 005 (3) * 0051 0010 (4) * Fluoride 011 001 (7) 012 002 (4) 0095 0012 (4) 011 001 (4) 0093 0009 (3) 017 002 (4) * Formate 025 001 (8) 045 004 (3) * 043 003 (3) * 035 004 (4) * 022 001 (3) 028 002 (3) Acetate 0090 0004 (8) 019 001 (3) * 018 001 (3) * 010 002 (5) 011 002 (3) 016 001 (3) * Propanoate 014 001 (3) 018 002 (4) 0098 0010 (4) * 0077 0013 (3) * 013 002 (3) Pyruvate 010 001 (5) 020 001 (3) * 013 002 (3) 0075 0015 (3) 017 003 (3) * Methane sulphonate 0077 0005 (5) 014 002 (4) * 0079 0014 (3) 0038 0004 (3) * 0088 0007 (3) Glutamate 0096 0008 (4) 0082 0009 (3) 0080 0008 (3) 0060 0012 (5) * 011 001 (3) Isethionate 013 001 (4) 011 001 (3) 0086 0012 (5) * 0043 0007 (3) * 0067 0005 (3) * Gluconate 0068 0004 (36) 010 001 (3) * 0060 0004 (3) 0044 0004 (3) 0077 0009 (3) 0088 0021 (5) Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.
Hanrahan Wild-type SCN > NO > Br > Cl > I > ClO > formate > F > PF T338A SCN > I NO > Br > ClO > Cl > PF > formate > F T338S SCN > NO I > Br > Cl > ClO > formate > PF > F T338N SCN > NO > Br > I = Cl > PF > ClO > formate > F T338V NO > SCN > Br = Cl > I > ClO > PF > formate > F J.
However, the permeabilities of the low conductance mutants T338N and T338V were more difficult to interpret, possibly indicating that substitution of a larger amino acid for T338 causes a more severe disruption of pore function.
In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0528 nm (wild-type), 0576 nm (T338A), 0549 nm (T338S), 0510 nm (T338N) and 0540 nm (T338V).
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0528 nm for wild-type, 0576 nm for T338A, 0549 nm for T338S, 0510 nm for T338N and 0540 nm for T338V.
In this case, fits by eqn (2) suggested minimum pore diameters of 0535 nm (wildtype), 0615 nm (T338A), 0505 nm (T338S), 0503 nm (T338N) and 0530 nm (T338V).
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0535 nm for wild type, 0615 nm for T338A, 0505 nm for T338S, 0503 nm for T338N and 0530 nm for T338V.
6), although the low conductances estimated for T338N, T338V and T338I should be considered rough approximations only.
All single channel conductances reported in this paper were measured at hyperpolarized potentials; conductance of the mutant channels T338N, T338V and T338I might be significantly lower at depolarized potentials (Fig.
I332K
protein
substitution
true positive
P13569
11B) and I332K (P.
T339V
protein
substitution
true positive
P13569
One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations.
In contrast, of five mutations made at T339, only one (T339V) produced detectable levels of CFTR protein in Western blots using cell lysates from one confluent 10 cm culture plate (approximately 2 10--3 10 cells; Fig.
Permeability of intracellular anions in wild-type and mutant CFTR Cl channels 7 Anion WT T338A T338S T338N T338V T339V Thiocyanate 263 013 (6) 585 027 (4) * 480 019 (3) * 872 103 (4) * 192 035 (4) * 328 008 (4) * Nitrate 153 004 (7) 204 008 (3) * 182 003 (4) * 422 022 (3) * 684 118 (7) * 161 002 (3) Bromide 123 003 (5) 174 004 (3) * 147 007 (3) * 166 015 (3) * 104 009 (5) 139 006 (4) * Chloride 100 001 (10) 100 002 (11) 100 002 (6) 100 003 (10) 100 004 (11) 100 006 (10) Iodide 084 003 (5) 209 016 (5) * 176 009 (3) * 103 005 (3) * 079 011 (3) 084 002 (3) Perchlorate 025 002 (6) 135 008 (3) * 066 006 (3) * 041 003 (3) * 054 000 (3) * 024 001 (4) Benzoate 0069 0006 (6) 017 003 (4) * 0091 0019 (3) 0089 0015 (4) 015 002 (4) * 0097 0014 (4) Hexafluorophosphate < 0019 (4) 053 001 (3) * 031 002 (3) * 068 002 (3) * 039 005 (3) * 0051 0010 (4) * Fluoride 011 001 (7) 012 002 (4) 0095 0012 (4) 011 001 (4) 0093 0009 (3) 017 002 (4) * Formate 025 001 (8) 045 004 (3) * 043 003 (3) * 035 004 (4) * 022 001 (3) 028 002 (3) Acetate 0090 0004 (8) 019 001 (3) * 018 001 (3) * 010 002 (5) 011 002 (3) 016 001 (3) * Propanoate 014 001 (3) 018 002 (4) 0098 0010 (4) * 0077 0013 (3) * 013 002 (3) Pyruvate 010 001 (5) 020 001 (3) * 013 002 (3) 0075 0015 (3) 017 003 (3) * Methane sulphonate 0077 0005 (5) 014 002 (4) * 0079 0014 (3) 0038 0004 (3) * 0088 0007 (3) Glutamate 0096 0008 (4) 0082 0009 (3) 0080 0008 (3) 0060 0012 (5) * 011 001 (3) Isethionate 013 001 (4) 011 001 (3) 0086 0012 (5) * 0043 0007 (3) * 0067 0005 (3) * Gluconate 0068 0004 (36) 010 001 (3) * 0060 0004 (3) 0044 0004 (3) 0077 0009 (3) 0088 0021 (5) Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.
Effects of mutations at T339 Of five amino acid substitutions carried out at position 339, only one (T339V) resulted in the production of detectable amounts of CFTR protein (Fig.
PKA- and ATP-dependent currents were observed, however, in twelve of fourteen T339V CFTR patches under the same conditions (Fig.
T339V showed slight inward current rectification under these symmetrical ionic conditions (-I + 50I-50 = 086 001; n = 8).
Mean chord conductances for T339V were 795 016 pS (n = 4) at -50 mV and 637 029 pS (n = 3) at +50 mV, compared with wild-type values of 791 009 pS (n = 18) at -50 mV and 785 009 pS (n = 12) at +50 mV.
As with T338A and T338S, T339V showed apparently normal channel gating, with open probability being time and Figure 9.
Furthermore, as with T338 mutants, T339V had negligible cation permeability (data not shown).
The T339V mutant also had significant alterations in ionic permeability (Fig.
Indeed, the selectivity sequence for T339V (SCN > NO > Br > Cl > I > ClO > formate > F > PF) was the same as for wild-type (see Table 2).
Gluconate permeability was not significantly altered in T339V, again suggesting no severe disruption of large organic anion permeability.
All of these effects are consistent with the T339V mutant having less severely altered pore properties than any of the T338 mutants studied.
Macroscopic current--voltage relationships for T339V CFTR anion selectivity of T339V, measured under biionic conditions with ClO or NO in the intracellular solution.
Note that the permeability of these ions in T339V is similar to that observed in wild-type (Fig.
A, T339V shows slight inward rectification with symmetrical Cl-containing solutions.
1993), T339V (Fig.
Unitary properties of T339V CFTR four to eighteen patches.
A, activity of a single T339V channel at -50 mV (cf.
4B and C) and T339V.
1C and D), or why the non-conservative T339V mutation alone should be appropriately processed (Fig.
Because of low protein expression, we were only able to characterize the permeation properties of one T339 mutant, T339V.
T339F
protein
substitution
true positive
P13569
Similar results were observed with T339A, T339Y and T339F (not shown).
Indeed, we found no PKA- and ATP-dependent currents in very large inside-out patches excised from BHK cells expressing T339A (n = 6), T339S (n = 4), T339Y (n = 6) or T339F (n = 5) using symmetrical 150 m NaCl-containing solutions (not shown).
T339S
protein
substitution
true positive
P13569
However, following immunoprecipitation of CFTR protein from five confluent plates, a very small amount of T339S mutant CFTR protein was detectable by Western blotting (Fig.
E, Western blot of immunoprecipitated T339S protein, carried out as described in Methods.
Indeed, we found no PKA- and ATP-dependent currents in very large inside-out patches excised from BHK cells expressing T339A (n = 6), T339S (n = 4), T339Y (n = 6) or T339F (n = 5) using symmetrical 150 m NaCl-containing solutions (not shown).
12021391
full text
S862A
protein
substitution
true negative
Whereas PKC phosphorylated the S873A, S877A, and S881A mutants as effectively as wild-type carboxyl-terminal fusion protein, phosphorylation of the S862A construct was markedly reduced (Fig.
Although the level of phosphorylation of the S873A, S877A, and S881A constructs by PKG were similar to the level of phosphorylation of wild-type ct-mGluR7, phosphorylation of the S862A ct-mGluR7 construct by PKG was markedly reduced (Fig.
Similar control responses to L-AP4 alone were seen for mutant receptors compared with wildtype receptors (1339 174 pA for wild-type; 1527 97 pA for the S862A mutant; n 9 to 11 for each).
Upon repeated application of L-AP4 after treatment with PMA, the inhibition of GIRK current activation was similar for the S862A receptor and wild-type receptor (Figs.
As seen for the wild-type receptor, the maximal effect of PMA on the S862A mutant receptor occurred after 12 min of PMA application (65 5 and 67 6% of control responses for wild-type and S862A receptors, respectively, n 9 to 11) and the inhibition of the mGluR7 S862A mutant receptor by PMA was significantly different from the effect of 4 -phorbol (Fig.
A, mutant full-length mGluR7 containing a single S862A point mutation was transfected into HEK cells stably expressing GIRK 1/2.
Control responses were 1418 223 pA for 4 -phorboltreated wild-type receptor, 1339 174 pA for the PMA-treated wild-type receptor, and 1527 97 pA for the S862A mutant receptor.
In the first, the ability of PKC to regulate the signaling of both a wild-type mGluR7 and mGluR7 S862A was determined.
Although PKC inhibits CaM binding to wild-type mGluR7, it has no effect on the ability of mGluR7 S862A to interact with CaM (Fig.
Although potential PKC-mediated effects directly at the level of the G protein or the GIRK channel itself provides a plausible explanation for the inhibition of mGluR7 (S862A) signaling, several lines of evidence argue against this.
S881A
protein
substitution
true negative
Whereas PKC phosphorylated the S873A, S877A, and S881A mutants as effectively as wild-type carboxyl-terminal fusion protein, phosphorylation of the S862A construct was markedly reduced (Fig.
Although the level of phosphorylation of the S873A, S877A, and S881A constructs by PKG were similar to the level of phosphorylation of wild-type ct-mGluR7, phosphorylation of the S862A ct-mGluR7 construct by PKG was markedly reduced (Fig.
S877A
protein
substitution
true negative
Whereas PKC phosphorylated the S873A, S877A, and S881A mutants as effectively as wild-type carboxyl-terminal fusion protein, phosphorylation of the S862A construct was markedly reduced (Fig.
Although the level of phosphorylation of the S873A, S877A, and S881A constructs by PKG were similar to the level of phosphorylation of wild-type ct-mGluR7, phosphorylation of the S862A ct-mGluR7 construct by PKG was markedly reduced (Fig.
S862E
protein
substitution
true negative
8C, application of L-AP4 to cells expressing the S862E mutant of mGluR7 activates GIRK current, and the signaling of this mutant is indistinguishable from signaling mediated by the wild-type receptor (maximal responses for wild-type 1nd S862E mutant receptors are 128 17 pA/pF and 131 a 9 pA/pF, respectively, n 9 to 10 for each).
C, HEK cells stably expressing GIRK 1/2 were transiently transfected with either wild-type mGluR7 or mGluR7 S862E cDNA.
Recordings from either wild-type (wt) (F) or S862E (E) transfected cells were combined and are mean S.E.
The second strategy used was to directly compare the functional response of a mutant mGluR7 that mimics the phosphorylated state and does not bind CaM, mGluR7 S862E, to wild-type mGluR7.
In the present study, mutation of a single serine residue (S862E) to disrupt CaM binding may allow for a more specific targeting of mGluR7/CaM interactions, thus providing a different view of the functional relevance of CaM binding to mGluR7 in HEK cells.
Recent studies indicate that the mGluR7 S862E point mutant also inhibits binding (El Far et al., 2001), which in theory could allow for enhanced r elease upon GTP binding to G protein subunits that offsets the effect of this mutation to disrupt CaM binding.
S873A
protein
substitution
true negative
Whereas PKC phosphorylated the S873A, S877A, and S881A mutants as effectively as wild-type carboxyl-terminal fusion protein, phosphorylation of the S862A construct was markedly reduced (Fig.
Although the level of phosphorylation of the S873A, S877A, and S881A constructs by PKG were similar to the level of phosphorylation of wild-type ct-mGluR7, phosphorylation of the S862A ct-mGluR7 construct by PKG was markedly reduced (Fig.
S317A
protein
substitution
true negative
The PKC-insensitive rat CB1 cannabinoid S317A mutant receptor (Garcia et al., 1998) was kindly provided by Dr.
To confirm that GIRK channels can be activated in the presence of PMA, a PKC-insensitive cannabinoid receptor (CB1 S317A) was transfected into the HEK cells.
Application of PMA to CB1 S317A-transfected HEK cells had no effect on the ability of a cannabinoid agonist to activate GIRK channels (Fig.
HEK cells stably expressing GIRK 1/2 were transiently transfected with CB1 S317A receptor cDNA and treated with 4 -phorbol or PMA as described in the legend to Fig.
Ken Mackie for providing the CB1 S317A cDNA.
M229R
protein
substitution
true negative
Rat mGluR7a in the pZEM229R vector (Saugstad et al., 1994) was digested with EcoRI and subcloned in-frame into the pTracer-EF/V5-His A vector.
11159439
full text
L164C
protein
substitution
true positive
Q14654
The mutant Kir6.2[L164C] is very sensitive to Cd2 block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP.
This pedestal is predicted to occur at 50 mM ATP in the L164C mutant, but at 1 mM in the double mutant L164C/R176A.
As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of 40%, with a clear plateau beyond 2 mM.
At a concentration that has no discernable inhibition of channel activity, ATP abolishes Cd2 sensitivity of Kir6.2[L164C] SUR1 (164C) channels.
In the double mutant Kir6.2[L164C, R176A], a clear plateau is observed in the maximum ATP inhibition.
RESULTS ATP prevents cadmium block and unblock in Kir6.2[L164C] SUR1 Residue 164 lines the channel pore in the M2 helix of the Kir6.2 subunit (Loussouarn et al., 2000); mutant Kir6.2[L164C] SUR1 (L164C) channels are inhibited by micromolar concentrations of cadmium, due to coordination by the thiol moieties of the cysteines at position 164 (Loussouarn et al., 2000).
L164C channels have a very high open probability with very low ATP sensitivity (Enkvetchakul et al., 2000; see Fig.
1 A demonstrates that 1 mM ATP r causes essentially no inhibition of L164C, but 10 M Cd2 eversibly blocks the channel.
In the same patch, despite no inhibitory effect of 1 mM ATP on L164C channels, the application of ATP before cadmium exposure completely prevents block by cadmium (Fig.
Biophysical Journal 80(2) 719 728 ATP Interaction with the KATP Channel Open State 721 FIGURE 1 ATP prevents cadmium block and unblock of Kir6.2[L164C] SUR1 (L164C) channels.
(A) Recording of a single patch with L164C exposed to 1 mM ATP or 10 M Cd2 as indicated; (B and C) Recording of L164C exposed to 10 M Cd2 alone, overlaid on recording of L164C exposed to both 10 M Cd2 and 1 mM ATP as indicated.
That ATP causes no inhibition of the channel but can prevent cadmium block and unblock indicates an action of ATP on the open state of L164C.
A plateau of ATP inhibition in L164C/R176A Wild-type KATP channels, formed of Kir6.2 SUR1 subunits, are very sensitive to ATP, being half-maximally inhibited at 10 M (Fig.
3 A, L164C is clearly very ATP insensitive and predicted to have a K1/2,ATP 100 mM (Fig.
1.7, K1/2 950 M, and C 0.64 (n Open-time distributions are altered by ATP in L164C/R176A Single-channel recordings of L164C/R176A were made in zero, 1, and 10 mM ATP (Figs.
FIGURE 3 ATP inhibition has a non-zero plateau in the high open-state stability mutant L164C/R176A.
(A and B) Representative recordings of L164C (A) and L164C/R176A (B) from inside-out membrane patches at 50 mV in K-INT solution, with application of ATP as indicated.
2), L164C (E), and L164C/R176A (F).
The mutation Kir6.2[R176A] has been shown to have a decreased PIP2 interaction (Shyng and Nichols, 1998; Fan and Makielski, 1999) and was added to the L164C mutation to create the double mutant Kir6.2[L164C, R176A] (L164C/R176A), to examine this possibility.
The L164C/R176A mutant expresses KATP channels that are still Cd2 sensitive (not shown) but which now have much higher apparent ATP sensitivity than L164C channels.
ATP inhibition was measured in the double mutant L164C/R176A at six different ATP concentrations (Fig.
ATP Interaction with the KATP Channel Open State 723 FIGURE 4 ATP alters open and closed time distributions in L164C/ R176A.
(A) Representative single-channel current of L164C/R176A recorded from a membrane patch on-cell and in inside-out mode at 50 mV membrane potential in symmetric K-INT solution.
FIGURE 5 Shifts in open and closed time distributions in L164C/R176A by ATP are reversible.
(A) Representative single-channel current of L164C/R176A recorded from another inside-out membrane patch at 50 mV membrane potential in symmetric K-INT solution.
At a concentration that has no discernible inhibition of channel activity, ATP abolishes Cd2 sensitivity of Kir6.2[L164C] SUR1 (164C) channels.
Accordingly, and in contrast to wild-type and low-open-state stability mutants, the L164C/R176A mutant shows two features of its behavior that provide direct evidence for ATP interaction with the open state, namely, a plateau in the ATP inhibition curve (Fig.
In the higher-open-state stability M158C and L164C mutations, this window becomes more prominent, and there is considerable steady-state occupancy at saturating [ATP].
By combining both stabilization of open states with respect to closed states (L164C or M158C), and destabilization of PIP2-bound states (Op, Cp) with respect to all other states (R176A), the double mutations result in a leftward shift of the ATP dose-response curve and a plateau of inhibition within the experimentally accessible range of [ATP].
4 and 5), as experimentally observed, although the multi-exponential nature of the L164C/R176A open-time distribution at high [ATP] is not particularly well reproduced.
The mutations L164C and M158C, both in the M2 domain, alter open-state stability by shifting all equilibria from closed to open states (indicated by asterisks), which then results in a significant occupancy of the ATP-bound open state.
inhibition, but a high fraction of ATP-bound open channels in L164C, at millimolar ATP.
Mutations within the M2 region (e.g., M158C and L164C) affect the relative stabilities of open and closed states.
Stabilization of the open state by these mutations, in particular by L164C, located at the lower end of M2, leads to increase in opening rate constants (asterisks) and significant, non-zero occupancy of the OA state in the presence of ATP.
c Recently, mutations in the voltage-gated Shaker K hannel P475D and P475E at the lower end of S6, analogous to the location of L164 at the lower end of M2, have been shown to have a non-zero minimum of open probability even at very negative membrane potentials (Hackos and Swartz, 2000), analogous to the pedestal seen with ATP in the L164C/R176A mutant.
*Rate constants from CP to OP, from C to O, and from CA to OA were all increased 125-fold, 625-fold, or 7500-fold for the increased open-state stability mutants (control C166S/N160D, M158C, and L164C, respectively).
Mutations M158C and L164C were then assumed to increase open-state stability by proportionally shifting equilibria between closed and open states by the same ratio (see Table 1).
Rate constants were adjusted with the above constraints to simulate single-channel openand closed-time distributions for wild type (data not shown) and for L164C/R176A (see Table 1).
P475E
protein
substitution
true positive
P08510
c Recently, mutations in the voltage-gated Shaker K hannel P475D and P475E at the lower end of S6, analogous to the location of L164 at the lower end of M2, have been shown to have a non-zero minimum of open probability even at very negative membrane potentials (Hackos and Swartz, 2000), analogous to the pedestal seen with ATP in the L164C/R176A mutant.
P475D
protein
substitution
true positive
P08510
c Recently, mutations in the voltage-gated Shaker K hannel P475D and P475E at the lower end of S6, analogous to the location of L164 at the lower end of M2, have been shown to have a non-zero minimum of open probability even at very negative membrane potentials (Hackos and Swartz, 2000), analogous to the pedestal seen with ATP in the L164C/R176A mutant.
N160D
protein
substitution
true positive
Q14654
Mutations in Kir6.2 were constructed in the background construct of Kir6.2[N160D, C166S] described previously (Loussouarn et al., 2000).
The N160D mutation allows measurement of zero current in the presence of the pore blocker spermine (Shyng et al., 1997a), and the C166S mutation removes a native cysteine that can react with cadmium.
FIGURE 2 ATP sensitivity of Kir6.2 SUR1 (wt) and of the background mutant Kir6.2[C166S, N160D] SUR1 (C166S/N160D) channels.
(C) [ATP]-response relationship of wt ( ) and C166S/N160D (f).
The mutations considered below were generated in the background of Kir6.2[C166S, N160D] (166S/160D).
Expression of C166S/N160D SUR1 generates channels that are considerably less sensitive to ATP than wild type, but which are nevertheless still apparently completely inhibited by high millimolar [ATP] (Fig.
The [ATP]-response curves for wild-type and C166S/N160D mutations (Fig.
(C) [ATP]-response relationships for the background C166S/N160D (f, taken from Fig.
For C166S/N160D mutant channels, there is a small window of [ATP] in which channels are predicted to be open with ATP bound.
*Rate constants from CP to OP, from C to O, and from CA to OA were all increased 125-fold, 625-fold, or 7500-fold for the increased open-state stability mutants (control C166S/N160D, M158C, and L164C, respectively).
M158C
protein
substitution
true positive
Q14654
ATP inhibition of M158C/R176A a measurable range, by decreasing the channel-phosphatidylinositol bisphosphate (PIP2) interaction.
ATP inhibition was measured in M158C/R176A, with leak currents measured in the presence of 20 M spermine at 50 mV membrane potential (Fig.
Steady-state [ATP]-response curves for M158C/R176A are fit by Eq.
FIGURE 6 A non-zero plateau in ATP inhibition is seen in the high open-state stability mutant M158C/R176A.
Representative recordings of M158C (A) and M158C/R176A (B) in inside-out patches at 50 mV membrane potential in K-INT solution, with application of ATP as indicated.
(C) ATP dose response of M158C (E) and M158C/R176A (F).
C166S
protein
substitution
true positive
Q14654
Mutations in Kir6.2 were constructed in the background construct of Kir6.2[N160D, C166S] described previously (Loussouarn et al., 2000).
The N160D mutation allows measurement of zero current in the presence of the pore blocker spermine (Shyng et al., 1997a), and the C166S mutation removes a native cysteine that can react with cadmium.
FIGURE 2 ATP sensitivity of Kir6.2 SUR1 (wt) and of the background mutant Kir6.2[C166S, N160D] SUR1 (C166S/N160D) channels.
(C) [ATP]-response relationship of wt ( ) and C166S/N160D (f).
The mutations considered below were generated in the background of Kir6.2[C166S, N160D] (166S/160D).
Expression of C166S/N160D SUR1 generates channels that are considerably less sensitive to ATP than wild type, but which are nevertheless still apparently completely inhibited by high millimolar [ATP] (Fig.
The [ATP]-response curves for wild-type and C166S/N160D mutations (Fig.
(C) [ATP]-response relationships for the background C166S/N160D (f, taken from Fig.
For C166S/N160D mutant channels, there is a small window of [ATP] in which channels are predicted to be open with ATP bound.
*Rate constants from CP to OP, from C to O, and from CA to OA were all increased 125-fold, 625-fold, or 7500-fold for the increased open-state stability mutants (control C166S/N160D, M158C, and L164C, respectively).
R176A
protein
substitution
true positive
Q14654
This pedestal is predicted to occur at 50 mM ATP in the L164C mutant, but at 1 mM in the double mutant L164C/R176A.
As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of 40%, with a clear plateau beyond 2 mM.
In the double mutant Kir6.2[L164C, R176A], a clear plateau is observed in the maximum ATP inhibition.
A plateau of ATP inhibition in L164C/R176A Wild-type KATP channels, formed of Kir6.2 SUR1 subunits, are very sensitive to ATP, being half-maximally inhibited at 10 M (Fig.
1.7, K1/2 950 M, and C 0.64 (n Open-time distributions are altered by ATP in L164C/R176A Single-channel recordings of L164C/R176A were made in zero, 1, and 10 mM ATP (Figs.
FIGURE 3 ATP inhibition has a non-zero plateau in the high open-state stability mutant L164C/R176A.
(A and B) Representative recordings of L164C (A) and L164C/R176A (B) from inside-out membrane patches at 50 mV in K-INT solution, with application of ATP as indicated.
2), L164C (E), and L164C/R176A (F).
ATP inhibition of M158C/R176A a measurable range, by decreasing the channel-phosphatidylinositol bisphosphate (PIP2) interaction.
The mutation Kir6.2[R176A] has been shown to have a decreased PIP2 interaction (Shyng and Nichols, 1998; Fan and Makielski, 1999) and was added to the L164C mutation to create the double mutant Kir6.2[L164C, R176A] (L164C/R176A), to examine this possibility.
The L164C/R176A mutant expresses KATP channels that are still Cd2 sensitive (not shown) but which now have much higher apparent ATP sensitivity than L164C channels.
ATP inhibition was measured in the double mutant L164C/R176A at six different ATP concentrations (Fig.
We again added the mutation R176A, predicted to shift the ATP dose-response curve to the left (see below) and allow estimation of the full dose-response relationship.
ATP inhibition was measured in M158C/R176A, with leak currents measured in the presence of 20 M spermine at 50 mV membrane potential (Fig.
Steady-state [ATP]-response curves for M158C/R176A are fit by Eq.
ATP Interaction with the KATP Channel Open State 723 FIGURE 4 ATP alters open and closed time distributions in L164C/ R176A.
(A) Representative single-channel current of L164C/R176A recorded from a membrane patch on-cell and in inside-out mode at 50 mV membrane potential in symmetric K-INT solution.
FIGURE 5 Shifts in open and closed time distributions in L164C/R176A by ATP are reversible.
(A) Representative single-channel current of L164C/R176A recorded from another inside-out membrane patch at 50 mV membrane potential in symmetric K-INT solution.
FIGURE 6 A non-zero plateau in ATP inhibition is seen in the high open-state stability mutant M158C/R176A.
Representative recordings of M158C (A) and M158C/R176A (B) in inside-out patches at 50 mV membrane potential in K-INT solution, with application of ATP as indicated.
(C) ATP dose response of M158C (E) and M158C/R176A (F).
Accordingly, and in contrast to wild-type and low-open-state stability mutants, the L164C/R176A mutant shows two features of its behavior that provide direct evidence for ATP interaction with the open state, namely, a plateau in the ATP inhibition curve (Fig.
Importantly, however, for both WT and R176A, states OA and CP are energetically so unfavorable that there is essentially no occupancy of these states at any [ATP], and the scheme can be simplified to model IIIa, in which ATP interacts only with the closed state, as in previous models (Shyng et al., 1997a; Enkvetchakul et al., 2000): Model IIIa Model II In this model, ATP can now be considered as an allosteric ligand that alters the equilibrium between open and closed states in each subunit.
By combining both stabilization of open states with respect to closed states (L164C or M158C), and destabilization of PIP2-bound states (Op, Cp) with respect to all other states (R176A), the double mutations result in a leftward shift of the ATP dose-response curve and a plateau of inhibition within the experimentally accessible range of [ATP].
4 and 5), as experimentally observed, although the multi-exponential nature of the L164C/R176A open-time distribution at high [ATP] is not particularly well reproduced.
Equilibrium constants (see Appendix) for wild type and for R176A were empirically selected to give K1/2,ATP of 10 M for both, and POzero of 0.6 for wild type and 0.1 for R176A.
The single assumption of the R176A mutation is a proportional decrease in the stability of the states CP and OP with respect 726 Enkvetchakul et al.
c Recently, mutations in the voltage-gated Shaker K hannel P475D and P475E at the lower end of S6, analogous to the location of L164 at the lower end of M2, have been shown to have a non-zero minimum of open probability even at very negative membrane potentials (Hackos and Swartz, 2000), analogous to the pedestal seen with ATP in the L164C/R176A mutant.
Rate constants from OP to O and from CP to C were increased 30-fold for the mutant R176A (which decreases PIP2 sensitivity).
Likewise, the mutation R176A was assumed to proportionally shift equilibria between PIP2-bound and free states by a certain ratio.
Rate constants were adjusted with the above constraints to simulate single-channel openand closed-time distributions for wild type (data not shown) and for L164C/R176A (see Table 1).
11328820
full text
N27A
protein
substitution
true negative
An enhanced association between PLB and SERCA2a would be consistent with the inability of isoproterenol to fully relieve the V49G mutant PLB superinhibitory effects, similar to previous findings with the N27A PLB mutant (24).
Previous studies on the N27A (24) and L37A and I40A (22) PLB superinhibitors also showed severely depressed SR and cardiac function in vivo, which were associated with hypertrophy by 3 months of age.
However, the hypertrophic phenotype of the N27A, L37A, and I40A mutant mice did not progress to overt heart failure, as observed with the V49G mutant.
V49G
protein
substitution
true negative
Kranias rom the Department of Pharmacology and Cell Biophysics, Division of Cardiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267, the Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, and the Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca2 transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val49 3 Gly mutant (2-fold), which is a superinhibitor of SR Ca2 -ATPase affinity for Ca2 , were generated, and their cardiac phenotype was examined longitudinally.
To further elucidate the functional significance of the PLB Val49 residue in vivo, we generated a mutant, Val49 3 Gly (V49G), which acts as a potent inhibitor of the Ca2 affinity of SERCA2a.
L37A
protein
substitution
true negative
Extension of these studies to in vivo models indicated that overexpression of either L37A or I40A mutant PLB resulted in depressed cardiac myocyte Ca2 kinetics and mechanical parameters associated with hypertrophy (22).
In contrast, the reduced contractility in L37A and I40A PLB mutant mice could be completely over come by isoproterenol stimulation (22).
Previous studies on the N27A (24) and L37A and I40A (22) PLB superinhibitors also showed severely depressed SR and cardiac function in vivo, which were associated with hypertrophy by 3 months of age.
However, the hypertrophic phenotype of the N27A, L37A, and I40A mutant mice did not progress to overt heart failure, as observed with the V49G mutant.
V49A
protein
substitution
true negative
Further support for the functional significance of the PLB transmembrane domain in mediating its regulatory effects on SERCA2a was provided by a recent study, in which a Val49 3 Ala (V49A) mutant PLB 1 24145 24146 Phospholamban and Contractile Failure .14 M, n 9), indicating a greater inhibitory effect compared with wild type PLB.
I40A
protein
substitution
true negative
Extension of these studies to in vivo models indicated that overexpression of either L37A or I40A mutant PLB resulted in depressed cardiac myocyte Ca2 kinetics and mechanical parameters associated with hypertrophy (22).
In contrast, the reduced contractility in L37A and I40A PLB mutant mice could be completely over come by isoproterenol stimulation (22).
Previous studies on the N27A (24) and L37A and I40A (22) PLB superinhibitors also showed severely depressed SR and cardiac function in vivo, which were associated with hypertrophy by 3 months of age.
However, the hypertrophic phenotype of the N27A, L37A, and I40A mutant mice did not progress to overt heart failure, as observed with the V49G mutant.
15272010
full text
C1400S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C1458S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C491S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C1410S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C590S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C866S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
C1355S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C524S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
M348C
protein
substitution
true positive
P13569
Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wildtype background.
TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment.
Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig.
2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers.
Mutant M348C/T1142C, for example, showed cross-linking with M5M and M8M but not with M17M.
It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12.
Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines.
Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig.
To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking.
Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C.
It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown).
To compare the inter-TMD interactions between WT and misprocessed CFTRs, the F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C.
6A, incorporation of the F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished crosslinking.
To test whether the lack of cross-linking in the F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking F508 mutation) would FIG.
To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 g/ml brefeldin A.
Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Time 0 is the start of stimulation by 10 M forskolin.
6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active.
The M348C/T1142C mutant showed a similar level of activity as WT CFTR.
T352C
protein
substitution
true negative
Reduced levels of the T352C and W356C mutants, however, were observed with M8M, and this may be due to aggregation.
Typo
T1142C
protein
substitution
true positive
P13569
Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wildtype background.
TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment.
Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig.
2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers.
Mutant M348C/T1142C, for example, showed cross-linking with M5M and M8M but not with M17M.
Mutant T351C/T1142C, on the other hand, shows extensive cross-linking with M8M but not with M5M or M17M.
It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12.
Therefore, it is not surprising that the substituted cysteines at both of these positions would crosslink to the same residue, T1142C.
Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines.
Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig.
To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking.
Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C.
It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown).
To compare the inter-TMD interactions between WT and misprocessed CFTRs, the F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C.
6A, incorporation of the F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished crosslinking.
To test whether the lack of cross-linking in the F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking F508 mutation) would FIG.
To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 g/ml brefeldin A.
Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Time 0 is the start of stimulation by 10 M forskolin.
6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active.
The M348C/T1142C mutant showed a similar level of activity as WT CFTR.
Both mutants T351C/T1142C and W356C/W1145C, however, exhibited 40% reduction in activity compared with WT CFTR.
L1143C
protein
substitution
true positive
P13569
An example of a mutant that did not show cross-linking, T351C/L1143C, is shown in Fig.
C1344S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
W1145C
protein
substitution
true positive
P13569
Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wildtype background.
TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment.
Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig.
2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers.
The positive mutant, W356C/ W1145C, showed cross-linking with all three cross-linkers (Fig.
Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines.
Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig.
To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking.
Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C.
It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown).
To compare the inter-TMD interactions between WT and misprocessed CFTRs, the F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C.
6A, incorporation of the F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished crosslinking.
To test whether the lack of cross-linking in the F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking F508 mutation) would FIG.
To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 g/ml brefeldin A.
Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Time 0 is the start of stimulation by 10 M forskolin.
6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active.
Both mutants T351C/T1142C and W356C/W1145C, however, exhibited 40% reduction in activity compared with WT CFTR.
C657S
protein
substitution
true negative
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Typo
W356C
protein
substitution
true positive
P13569
Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wildtype background.
TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment.
Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig.
2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers.
The positive mutant, W356C/ W1145C, showed cross-linking with all three cross-linkers (Fig.
Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines.
Reduced levels of the T352C and W356C mutants, however, were observed with M8M, and this may be due to aggregation.
Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig.
To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking.
Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C.
It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown).
To compare the inter-TMD interactions between WT and misprocessed CFTRs, the F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C.
6A, incorporation of the F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished crosslinking.
To test whether the lack of cross-linking in the F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking F508 mutation) would FIG.
To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 g/ml brefeldin A.
Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Time 0 is the start of stimulation by 10 M forskolin.
6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active.
Both mutants T351C/T1142C and W356C/W1145C, however, exhibited 40% reduction in activity compared with WT CFTR.
C832S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C343S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
C225S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C592S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
T351C
protein
substitution
true positive
P13569
Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wildtype background.
TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment.
Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig.
2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers.
Mutant T351C/T1142C, on the other hand, shows extensive cross-linking with M8M but not with M5M or M17M.
It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12.
An example of a mutant that did not show cross-linking, T351C/L1143C, is shown in Fig.
Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines.
Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig.
To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking.
Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C.
It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown).
To compare the inter-TMD interactions between WT and misprocessed CFTRs, the F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C.
6A, incorporation of the F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished crosslinking.
To test whether the lack of cross-linking in the F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking F508 mutation) would FIG.
To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 g/ml brefeldin A.
Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Time 0 is the start of stimulation by 10 M forskolin.
6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active.
Both mutants T351C/T1142C and W356C/W1145C, however, exhibited 40% reduction in activity compared with WT CFTR.
C1395S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
C76S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
C126S
protein
substitution
true negative
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
C276S
protein
substitution
true positive
P13569
The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments.
Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21.
10086971
full text
S818L
protein
substitution
true positive
Q12809
Methods and Results--New specific primers allowed the amplification of the 3 part of HERG, the identification of 2 missense mutations, S818L and V822 M, in the putative cyclic nucleotide binding domain, and a 1-bp insertion, 3108 1G.
b, Partial HERG sequences of mutation carriers: a C to T substitution at position 2453 (S818L) is observed in Family 10025 and a G to A substitution at position 2464 (V822 M) in Family 10626.
We identified 2 missense mutations, the mutation V822 M, previously described by Satler and coworkers, in a large Irish family,19 and another missense mutation very close to the previous one, S818L.
R555C
protein
substitution
true positive
P51787
In KCNQ1, 2 missense mutations, R539W and R555C, were found in Romano-Ward families, 16,17 in addition to an insertion-deletion in Jervell and Lange-Nielsen patients.18 In HERG, a splicing mutation,4 a missense mutation, V822 M, in the putative nucleotide binding site,19 and a 31-bp duplication were identified in the 3 part of the gene.20 This suggests that the C-terminal parts of these genes should also be analyzed for disease-causing mutations.
Electrophysiological characterization of these HERG mutations would shed light on their molecular mechanisms and may allow prediction of clinical outcome, as has been attempted for the KCNQ1 C-terminal mutation, R555C.16,17 In contrast, in Family 5969, the fourth HERG mutation we identified, 2592 1G3 A, is a substitution which disrupts the splice-donor sequence of intron 10 and appears more severe because 2 sudden deaths occurred at the ages of 9 and 20, and one of the 2 living carriers had syncopes at 4 years with a 2-day coma.
A341E
protein
substitution
true positive
P51787
Lastly, in a large family, a maternally inherited G to A transition was found in the splicing donor consensus site of HERG, 2592 1G-A, and a paternally inherited mutation, A341E, was identified in KCNQ1.
The KCNQ1 A341E mutation causes the loss of Hha I enzymatic site: the control individual (C) and the unaffected individuals present a normal pattern with 2 fragments (174 and 73 bp).
The paternally inherited mutation, A341E, was the most frequently identified KCNQ1 mutation.
R539W
protein
substitution
true positive
P51787
In KCNQ1, 2 missense mutations, R539W and R555C, were found in Romano-Ward families, 16,17 in addition to an insertion-deletion in Jervell and Lange-Nielsen patients.18 In HERG, a splicing mutation,4 a missense mutation, V822 M, in the putative nucleotide binding site,19 and a 31-bp duplication were identified in the 3 part of the gene.20 This suggests that the C-terminal parts of these genes should also be analyzed for disease-causing mutations.
V822M
protein
substitution
true positive
Q12809
Methods and Results--New specific primers allowed the amplification of the 3 part of HERG, the identification of 2 missense mutations, S818L and V822 M, in the putative cyclic nucleotide binding domain, and a 1-bp insertion, 3108 1G.
In KCNQ1, 2 missense mutations, R539W and R555C, were found in Romano-Ward families, 16,17 in addition to an insertion-deletion in Jervell and Lange-Nielsen patients.18 In HERG, a splicing mutation,4 a missense mutation, V822 M, in the putative nucleotide binding site,19 and a 31-bp duplication were identified in the 3 part of the gene.20 This suggests that the C-terminal parts of these genes should also be analyzed for disease-causing mutations.
b, Partial HERG sequences of mutation carriers: a C to T substitution at position 2453 (S818L) is observed in Family 10025 and a G to A substitution at position 2464 (V822 M) in Family 10626.
We identified 2 missense mutations, the mutation V822 M, previously described by Satler and coworkers, in a large Irish family,19 and another missense mutation very close to the previous one, S818L.
In the large 4 generation Irish family with the V822 M mutation (described by Satler and coworkers), 16 members were considered clinically affected, on the basis of electrocardiographic and clinical criteria, but 26 were found to be genetically affected by linkage and SSCP analysis.19 No history of sudden death was reported in this family.
The majority of the V822 M carriers was asymptomatic (19 of 26, 73%).
11401822
full text
E571R
protein
substitution
P37088
true positive
In that study, reversing the negative charges of the three amino acids of the M2 region (E568R, E571R, and D575R) significantly decreased the channel conductance without affecting ion selectivity.
D575R
protein
substitution
P37088
true positive
In that study, reversing the negative charges of the three amino acids of the M2 region (E568R, E571R, and D575R) significantly decreased the channel conductance without affecting ion selectivity.
E108R
protein
substitution
P37088
true positive
In contrast, similar mutation (E108R) of the M1 region of -hENaC showed no functional consequences (10).
E568R
protein
substitution
P37088
true positive
In that study, reversing the negative charges of the three amino acids of the M2 region (E568R, E571R, and D575R) significantly decreased the channel conductance without affecting ion selectivity.
11095943
full text
L1014F
protein
substitution
true negative
3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS position identical to the k d r allele L1014F in house flies; mutation at this position led to the first recorded incidence of k d r resistance (4) and is now recognized as the most common among k d r -like pyrethroid-resistant insect species (18 20).
Recent publications have demonstrated the role of L1014F in conferring reduced sodium channel sensitivity to the pyrethroids cismethrin and deltamethrin (21, 22).
This phenomenon also was observed to result from the homologous II-S6 mutation (L1014F) in kdr houseflies (21, 22).
L1029H
protein
substitution
true negative
Pyrethroid resistant strains of the moth Heliothis virescens carry single point mutations leading to amino acid substitutions in either transmembrane segment I-S6 (V421M) or II-S6 (L1029H) of the para-homologous sodium channel.
We analyzed the consequences of V421M and L1029H mutations constructed in the Drosophila para sodium channel heterologously expressed in Xenopus oocytes, and found that both mutations confer channel insensitivity to permethrin, with the L1029H mutation having a more pronounced effect.
Both mutations also modify the intrinsic voltage-dependent gating properties of the channel, but L1029H less so than V421M.
These results suggest that mutation V421M exacts a higher fitness cost than L1029H, providing a plausible explanation for genetic succession observed in field strains, where V421M was replaced by L1029H during the past decade.
A single point mutation in transmembrane segment I-S6 involves substitution of methionine for valine (V421M), while the other mutation substitutes histidine for leucine (L1029H) in II-S6 (9 11).
This work suggests that the mutations V421M and L1029H confer pyrethroid insensitivity, but other variables affecting channel regulation can only be ruled out upon further analysis of the mutations in a defined, heterologously expressed sodium channel.
(A) Primary structure of the sodium channel, showing two point mutations (V421M and L1029H), each identified from different pyrethroid-resistant strains of the moth, H.
The primers for V421M mutation were 5 -GGTTCATTCTATCTTATGAATTTGATTTTGGCC and 5 -GGCCAAAATCAAATTCATAAGATAGAATGAACC, and for L1029H mutation were 5 -GGTTAAGTACCACATGATTGCCGATGACAACGG and 5 -CCGTTGTCATCGGCAATCATGTGGTACTTAACC.
Both V421M and L1029H mutations reduced sodium channel sensitivity to permethrin, with L1029H having a more pronounced effect.
The highest concentration of permethrin tested (10 M) modified 16.5 2.9% of wild-type channels, but modified only 7.2 0.8% of channels carrying the mutation L421M and 4.1 0.5% of channels carrying the L1029H mutation (Fig.
These results show that the L1029H mutation has a greater protective effect against pyrethroid poisoning than the V421M mutation.
tance, 50% activation of wild-type channels was shifted in the positive direction by 6.6 mV and 3.3 mV for the V421M and L1029H mutant channels, respectively (Fig.
The V421M mutation thus shifts voltage-dependent activation approximately twice as much as the L1029H mutation, and indicates that channels carrying resistance mutations require more positive potentials for gating to occur.
Steady-state inactivation curves for V421M and L1029H mutant channels also were shifted in the positive direction compared to wild-type (Fig.
Prepulses required for 50% sodium channel inactivation of V421M and L1029H mutant channels were shifted in the positive direction by 4.5 mV and 2.1 mV, respectively.
Again, the V421M mutation produced a more pronounced shift in gating than the L1029H mutation.
Permethrin-modified tail currents recorded from the wild type para sodium channel compared to the V421M (A) and L1029H (B) mutant channels.
Voltage dependent activation (A) and steady-state inactivation (B) of wild-type, V421M, and L1029H sodium channels.
Average time constants obtained were 9044 540 ms (wild-type; n 7), 174 4 81 ms (V421M; n 10) and 158 35 ms (L1029H; n ) (Figs.
DISCUSSION The point mutations V421M and L1029H, each linked to pyrethroid resistance in H.
The L1029H mutation occurs at a modified channels.
4) and subsequently decayed according to a single exponential TABLE 1 Voltage-Dependent Activation and Steady-State Inactivation of Wild-Type, V421M, and L1029H Sodium Channels Insect strains Wild-type V421M L1029H Voltage dependent activation, half-maximal conductance potential (mV, SD) 27.2 20.6 23.9 3.2 (n 3.4 (n 2.5 (n 15) a 15) b 16) b a Steady-state inactivation, half inactivation potential (mV, SD) 47.7 43.2 45.6 1.3 (n 2.7 (n 2.7 (n 0.05).
virescens carrying either the V421M or L1029H mutation (12, 13).
This work unambiguously shows that reduced pyrethroid sensitivity occurs in a cloned sodium channel carrying either V421M or L1029H mutations.
We note in particular that the L1029H mutation is more effective in reducing permethrin sensitivity than the V421M mutation.
Consequences of Compromised Channel Function Both the V421M and L1029H mutations modify normal gating properties of the sodium channel by shifting voltage-dependent activation to more positive potentials.
Even though the V421M and L1029H mutations may confer advantages during pyrethroid exposure, concomitant changes in gating properties led to decreased excitability and sluggish behavior observed in our earlier studies.
Our quantitative comparisons show that the L1029H mutation is more effective in reducing permethrin sensitivity, yet less intrusive in compromising channel gating function than the V421M mutation.
What implications might this have on the relative efficacy of each mutation in the overall resistance phenomenon? Population genetic analysis of the V421M and L1029H mutations in H.
virescens showed that V421M appeared early as multiple mutational events, whereas L1029H arose more recently as a single mutational event that has remained stable in resistant populations (25).
virescens indicate that L1029H replaced V421M during the last ten years (25).
Specifically, the frequencies of V421M and L1029H in 1990 were 0.21 and 0.2, respectively (n 128).
In 1996 and 1997 (n 49 and 142, respectively), the frequency of L1029H rose to 0.76 and 0.78, while the frequency of V421M dropped below detectable levels.
Some Effects on Channel Gating May be Adaptive A second effect of the V421M and L1029H mutations on channel gating is an increased rate of deactivation observed in permethrin-modified channels.
In summary, we have demonstrated that the mutations V421M and L1029H reduce sodium channel sensitivity to permethrin and thus provide a structural basis for pyrethroid resistance.
The survival advantage gained in resistance to pyrethroids outweighs compromised channel function, particularly in the case of the L1029H mutation.
The relative efficiency of such point mutations as protective factors has been illustrated by replacement of V421M by L1029H in pest insect populations during the 1990s, representing an episode of genetic succession (25).
V421M
protein
substitution
true negative
Pyrethroid resistant strains of the moth Heliothis virescens carry single point mutations leading to amino acid substitutions in either transmembrane segment I-S6 (V421M) or II-S6 (L1029H) of the para-homologous sodium channel.
We analyzed the consequences of V421M and L1029H mutations constructed in the Drosophila para sodium channel heterologously expressed in Xenopus oocytes, and found that both mutations confer channel insensitivity to permethrin, with the L1029H mutation having a more pronounced effect.
Both mutations also modify the intrinsic voltage-dependent gating properties of the channel, but L1029H less so than V421M.
These results suggest that mutation V421M exacts a higher fitness cost than L1029H, providing a plausible explanation for genetic succession observed in field strains, where V421M was replaced by L1029H during the past decade.
A single point mutation in transmembrane segment I-S6 involves substitution of methionine for valine (V421M), while the other mutation substitutes histidine for leucine (L1029H) in II-S6 (9 11).
This work suggests that the mutations V421M and L1029H confer pyrethroid insensitivity, but other variables affecting channel regulation can only be ruled out upon further analysis of the mutations in a defined, heterologously expressed sodium channel.
(A) Primary structure of the sodium channel, showing two point mutations (V421M and L1029H), each identified from different pyrethroid-resistant strains of the moth, H.
The primers for V421M mutation were 5 -GGTTCATTCTATCTTATGAATTTGATTTTGGCC and 5 -GGCCAAAATCAAATTCATAAGATAGAATGAACC, and for L1029H mutation were 5 -GGTTAAGTACCACATGATTGCCGATGACAACGG and 5 -CCGTTGTCATCGGCAATCATGTGGTACTTAACC.
Both V421M and L1029H mutations reduced sodium channel sensitivity to permethrin, with L1029H having a more pronounced effect.
These results show that the L1029H mutation has a greater protective effect against pyrethroid poisoning than the V421M mutation.
Oocytes expressing wild-type (A) and V421M (B) were depolarized with test potentials to 10 mV at 10 s intervals (holding potential, 100 mV) following exposure to increasing concentrations of permethrin.
tance, 50% activation of wild-type channels was shifted in the positive direction by 6.6 mV and 3.3 mV for the V421M and L1029H mutant channels, respectively (Fig.
The V421M mutation thus shifts voltage-dependent activation approximately twice as much as the L1029H mutation, and indicates that channels carrying resistance mutations require more positive potentials for gating to occur.
Steady-state inactivation curves for V421M and L1029H mutant channels also were shifted in the positive direction compared to wild-type (Fig.
Prepulses required for 50% sodium channel inactivation of V421M and L1029H mutant channels were shifted in the positive direction by 4.5 mV and 2.1 mV, respectively.
Again, the V421M mutation produced a more pronounced shift in gating than the L1029H mutation.
Permethrin-modified tail currents recorded from the wild type para sodium channel compared to the V421M (A) and L1029H (B) mutant channels.
Voltage dependent activation (A) and steady-state inactivation (B) of wild-type, V421M, and L1029H sodium channels.
Average time constants obtained were 9044 540 ms (wild-type; n 7), 174 4 81 ms (V421M; n 10) and 158 35 ms (L1029H; n ) (Figs.
DISCUSSION The point mutations V421M and L1029H, each linked to pyrethroid resistance in H.
4) and subsequently decayed according to a single exponential TABLE 1 Voltage-Dependent Activation and Steady-State Inactivation of Wild-Type, V421M, and L1029H Sodium Channels Insect strains Wild-type V421M L1029H Voltage dependent activation, half-maximal conductance potential (mV, SD) 27.2 20.6 23.9 3.2 (n 3.4 (n 2.5 (n 15) a 15) b 16) b a Steady-state inactivation, half inactivation potential (mV, SD) 47.7 43.2 45.6 1.3 (n 2.7 (n 2.7 (n 0.05).
The mutation V421M, which occurs in the homologous transmembrane segment S6 in domain I, is a unique structural alteration associated with pyrethroid resistance in H.
virescens carrying either the V421M or L1029H mutation (12, 13).
This work unambiguously shows that reduced pyrethroid sensitivity occurs in a cloned sodium channel carrying either V421M or L1029H mutations.
We note in particular that the L1029H mutation is more effective in reducing permethrin sensitivity than the V421M mutation.
Consequences of Compromised Channel Function Both the V421M and L1029H mutations modify normal gating properties of the sodium channel by shifting voltage-dependent activation to more positive potentials.
Our earlier study showed that voltage-dependent activation of sodium channels in central neurons of moths carrying the V421M mutation was shifted 14 mV in the positive direction, which coincided with reduced neuronal excitability and markedly sluggish behavior (12).
Even though the V421M and L1029H mutations may confer advantages during pyrethroid exposure, concomitant changes in gating properties led to decreased excitability and sluggish behavior observed in our earlier studies.
virescens carrying the V421M mutation (23).
Our quantitative comparisons show that the L1029H mutation is more effective in reducing permethrin sensitivity, yet less intrusive in compromising channel gating function than the V421M mutation.
What implications might this have on the relative efficacy of each mutation in the overall resistance phenomenon? Population genetic analysis of the V421M and L1029H mutations in H.
virescens showed that V421M appeared early as multiple mutational events, whereas L1029H arose more recently as a single mutational event that has remained stable in resistant populations (25).
virescens indicate that L1029H replaced V421M during the last ten years (25).
Specifically, the frequencies of V421M and L1029H in 1990 were 0.21 and 0.2, respectively (n 128).
In 1996 and 1997 (n 49 and 142, respectively), the frequency of L1029H rose to 0.76 and 0.78, while the frequency of V421M dropped below detectable levels.
The neurophysiological and pharmacological analyses reported here demonstrate that the V421M mutation involves a higher fitness cost due to altered channel gating properties, and provide cellular and molecular bases for the evolutionary succession of mutations observed in field populations during the 1990s.
Some Effects on Channel Gating May be Adaptive A second effect of the V421M and L1029H mutations on channel gating is an increased rate of deactivation observed in permethrin-modified channels.
In summary, we have demonstrated that the mutations V421M and L1029H reduce sodium channel sensitivity to permethrin and thus provide a structural basis for pyrethroid resistance.
The relative efficiency of such point mutations as protective factors has been illustrated by replacement of V421M by L1029H in pest insect populations during the 1990s, representing an episode of genetic succession (25).
L421M
protein
substitution
true negative
The highest concentration of permethrin tested (10 M) modified 16.5 2.9% of wild-type channels, but modified only 7.2 0.8% of channels carrying the mutation L421M and 4.1 0.5% of channels carrying the L1029H mutation (Fig.
10318798
full text
P625A
protein
substitution
true positive
P37091
Image mutation
P626A
protein
substitution
true positive
P37091
Image mutation
P565X
protein
substitution
P37090
true positive
When expressed in Xenopus oocytes, most of the ENaC stop ( -H647X, -P565X, -S608X) or point ( -P671A, -Y618A, -P(624 626)A) mutations induced enhanced Na currents when compared with wild type , , -rENaC.
For the C-terminal stop mutations -H647X, -P565X, -S608X the following sense (s) and antisense (as) oligonucleotides (5 -3 ) were used: -H647X, CGACCCACGCGTCGCG (s); AGGACAGAAACGGGACG (as); -P565X, CCCACGCGTCCGACC (s), GCCGCCTCCTGCGCA (as); -S608X, TCGACCCACGCGTCC (s), AGGTAAAAGTGGGCAGGTC (as).
Representative examples of whole cell currents and inhibition by amiloride (10 mol/l) observed in oocytes expressing , , -rENaC (upper trace) or , -P565X, -rENaC (lower trace).
RESULTS Effects of Deletion of C-terminal PY Motifs in , , rENaC--PY motifs in either -, -, or -subunits of rENaC were deleted by either C-terminal point ( -P671A, -Y618A, -P(624 626)A) or C-terminal stop mutations ( -H647X, -P565X, -S608X).
A representative record of the whole cell current from an oocyte coexpressing -P565X with wild type and -subunits is shown in the lower trace of Fig.
2 GENaC was significantly enhanced for -P565X, -S608X, -P671A, and -Y618A, while -H647X and -P(624 626)A did not produce enhanced conductances.
S608X
protein
substitution
P37091
true positive
When expressed in Xenopus oocytes, most of the ENaC stop ( -H647X, -P565X, -S608X) or point ( -P671A, -Y618A, -P(624 626)A) mutations induced enhanced Na currents when compared with wild type , , -rENaC.
For the C-terminal stop mutations -H647X, -P565X, -S608X the following sense (s) and antisense (as) oligonucleotides (5 -3 ) were used: -H647X, CGACCCACGCGTCGCG (s); AGGACAGAAACGGGACG (as); -P565X, CCCACGCGTCCGACC (s), GCCGCCTCCTGCGCA (as); -S608X, TCGACCCACGCGTCC (s), AGGTAAAAGTGGGCAGGTC (as).
RESULTS Effects of Deletion of C-terminal PY Motifs in , , rENaC--PY motifs in either -, -, or -subunits of rENaC were deleted by either C-terminal point ( -P671A, -Y618A, -P(624 626)A) or C-terminal stop mutations ( -H647X, -P565X, -S608X).
2 GENaC was significantly enhanced for -P565X, -S608X, -P671A, and -Y618A, while -H647X and -P(624 626)A did not produce enhanced conductances.
Similar to , , rENaC, also , , -S608X-rENaC whole cell conductances were down-regulated upon stimulation of CFTR with IBMX and forskolin (Fig.
Inhibition of , , -rENaC (A) and , , S608X-rENaC (B) by CFTR.
The effects of amiloride on both wt , , -rENaC and , , S608X-rENaC were attenuated after activation of a CFTR Cl conductance by IBMX (1 mmol/l) and forskolin (IBMX/Fors, 10 mol/l).
T592M
protein
substitution
P37090
true positive
Another mutant, -T592M,T593A-ENaC, also showed enhanced Na currents, which were down-regulated by CFTR.
MATERIALS AND METHODS Liddle and T592M,T593A Mutations--Mutations of the rat epithelial Na channel subunits were generated by polymerase chain reaction.
T592M,T593A was created using the oligonucleotides TCTTCCAGCCTGACATGGCTAGCTGCAGGCCCAAT (s), ATTGGGCCTGCAGCTAGCCATGTCAGGCTGGAA (as).
CFTR-dependent Inhibition of T592M,T593A-ENaC--According to previous reports, a mutation in the -subunit of the human ENaC ( -T594M) causes a loss of protein kinase C inhibition and leads to salt-sensitive hypertension in the African-American population (23).
T592M,T593A-rENaC, when coexpressed with equal amounts of - and -subunits, led to an amiloride sensitive Na conductance that was significantly higher than in oocytes from the same batch injected with wt , , -ENaC (Fig.
Stimulation with IBMX and forskolin had no effect on , T592M,T593A, -rENaC Na conductance (13.7 1.2 versus 13.5 1.9 microsiemens; n 4).
When coexpressed with CFTR, , T592M,T593A, -rENaC conductance was significantly attenuated by stimulation with IBMX and forskolin (Fig.
Similar holds true for T592M,T593A-ENaC and ENaC currents activated by proteases.
This also applies to other gain of function mutations like T592M,T593A-rENaC, which was not regulated directly by cAMP in our hands (23).
T594M
protein
substitution
P51168
true positive
CFTR-dependent Inhibition of T592M,T593A-ENaC--According to previous reports, a mutation in the -subunit of the human ENaC ( -T594M) causes a loss of protein kinase C inhibition and leads to salt-sensitive hypertension in the African-American population (23).
CFTR-dependent inhibition of the ENaC mutant T593A/T594M.
P624A
protein
substitution
true positive
P37091
Image mutation
H647X
protein
substitution
true positive
P37089
When expressed in Xenopus oocytes, most of the ENaC stop ( -H647X, -P565X, -S608X) or point ( -P671A, -Y618A, -P(624 626)A) mutations induced enhanced Na currents when compared with wild type , , -rENaC.
For the C-terminal stop mutations -H647X, -P565X, -S608X the following sense (s) and antisense (as) oligonucleotides (5 -3 ) were used: -H647X, CGACCCACGCGTCGCG (s); AGGACAGAAACGGGACG (as); -P565X, CCCACGCGTCCGACC (s), GCCGCCTCCTGCGCA (as); -S608X, TCGACCCACGCGTCC (s), AGGTAAAAGTGGGCAGGTC (as).
RESULTS Effects of Deletion of C-terminal PY Motifs in , , rENaC--PY motifs in either -, -, or -subunits of rENaC were deleted by either C-terminal point ( -P671A, -Y618A, -P(624 626)A) or C-terminal stop mutations ( -H647X, -P565X, -S608X).
2 GENaC was significantly enhanced for -P565X, -S608X, -P671A, and -Y618A, while -H647X and -P(624 626)A did not produce enhanced conductances.
T593A
protein
substitution
P37090
true positive
Another mutant, -T592M,T593A-ENaC, also showed enhanced Na currents, which were down-regulated by CFTR.
MATERIALS AND METHODS Liddle and T592M,T593A Mutations--Mutations of the rat epithelial Na channel subunits were generated by polymerase chain reaction.
T592M,T593A was created using the oligonucleotides TCTTCCAGCCTGACATGGCTAGCTGCAGGCCCAAT (s), ATTGGGCCTGCAGCTAGCCATGTCAGGCTGGAA (as).
CFTR-dependent Inhibition of T592M,T593A-ENaC--According to previous reports, a mutation in the -subunit of the human ENaC ( -T594M) causes a loss of protein kinase C inhibition and leads to salt-sensitive hypertension in the African-American population (23).
T592M,T593A-rENaC, when coexpressed with equal amounts of - and -subunits, led to an amiloride sensitive Na conductance that was significantly higher than in oocytes from the same batch injected with wt , , -ENaC (Fig.
Stimulation with IBMX and forskolin had no effect on , T592M,T593A, -rENaC Na conductance (13.7 1.2 versus 13.5 1.9 microsiemens; n 4).
When coexpressed with CFTR, , T592M,T593A, -rENaC conductance was significantly attenuated by stimulation with IBMX and forskolin (Fig.
CFTR-dependent inhibition of the ENaC mutant T593A/T594M.
B, summary of GENaC produced by wild type , , -ENaC or , -T593A/594M, ENaC.
C, summary of GENaC produced by , -T593A/594M, -ENaC before (white bar) and after (black bar) stimulation with IBMX and forskolin (IBMX/Fors).
Similar holds true for T592M,T593A-ENaC and ENaC currents activated by proteases.
This also applies to other gain of function mutations like T592M,T593A-rENaC, which was not regulated directly by cAMP in our hands (23).
Y618A
protein
substitution
P37090
true positive
When expressed in Xenopus oocytes, most of the ENaC stop ( -H647X, -P565X, -S608X) or point ( -P671A, -Y618A, -P(624 626)A) mutations induced enhanced Na currents when compared with wild type , , -rENaC.
C-terminal point mutations -P671A, -Y618A, -P (624 626)A were generated using GACAGCCCCTCCAGCTGCCTATGCTACT (s), AGTAGCATAGCCAGCTGGAGGG-GCTGTC (as) for -P671A); GCACTCCACCTCCCAATGCGCACTCCCTGAGGCTG (s), CAGCCTCAGGGAGTGGCGATTGGGAGGTGGAGTGC (as) for -Y618A; GGTGCCTGGCACAGCGGCCGCCAGATACAATA (s), TATTGTATCTGGCGGCCGCTGTGCCTGGTATT (as) for -P (624 626)A).
RESULTS Effects of Deletion of C-terminal PY Motifs in , , rENaC--PY motifs in either -, -, or -subunits of rENaC were deleted by either C-terminal point ( -P671A, -Y618A, -P(624 626)A) or C-terminal stop mutations ( -H647X, -P565X, -S608X).
2 GENaC was significantly enhanced for -P565X, -S608X, -P671A, and -Y618A, while -H647X and -P(624 626)A did not produce enhanced conductances.
P671A
protein
substitution
true positive
P37089
When expressed in Xenopus oocytes, most of the ENaC stop ( -H647X, -P565X, -S608X) or point ( -P671A, -Y618A, -P(624 626)A) mutations induced enhanced Na currents when compared with wild type , , -rENaC.
C-terminal point mutations -P671A, -Y618A, -P (624 626)A were generated using GACAGCCCCTCCAGCTGCCTATGCTACT (s), AGTAGCATAGCCAGCTGGAGGG-GCTGTC (as) for -P671A); GCACTCCACCTCCCAATGCGCACTCCCTGAGGCTG (s), CAGCCTCAGGGAGTGGCGATTGGGAGGTGGAGTGC (as) for -Y618A; GGTGCCTGGCACAGCGGCCGCCAGATACAATA (s), TATTGTATCTGGCGGCCGCTGTGCCTGGTATT (as) for -P (624 626)A).
RESULTS Effects of Deletion of C-terminal PY Motifs in , , rENaC--PY motifs in either -, -, or -subunits of rENaC were deleted by either C-terminal point ( -P671A, -Y618A, -P(624 626)A) or C-terminal stop mutations ( -H647X, -P565X, -S608X).
2 GENaC was significantly enhanced for -P565X, -S608X, -P671A, and -Y618A, while -H647X and -P(624 626)A did not produce enhanced conductances.
12589089
full text
C1573T
protein
substitution
true negative
The first mutation (C1573T) resulted N TACCGAGTG Tyr Arg Val F TACNGAGTG Tyr C T Arg Stop Val M TACCGAGTG Tyr Arg Val P TACNGAGTG Tyr C T Arg Stop Val A N GCCATGTGC Ala Met Cys F GCCATGTGC Ala Met Cys M GCCANGTGC Ala T C Met Thr Cys P GCCANGTGC Ala T C Met Thr Cys B Fig.
R338STOP
protein
substitution
true positive
P48048
We have identified amino acid exchanges Arg338Stop and Met357Thr in the gene exon 5 for ROMK by PCR and direct sequencing.
(A) Corresponding DNA sequence (sense strand) showing the Arg338Stop mutation; (B) Corresponding DNA sequence (sense strand) showing the Met357Thr mutation.
The Arg338Stop and Met357Thr mutations located in the C-terminal intracellular part were transmitted from the father and mother of the patient, respectively, thereby giving rise to a compound heterozygote mutation in the patient.
Mutational analysis of ROMK gene in this patient with antenatal Bartter syndrome showing interesting clinical manifestations revealed two mutations Arg338Stop and Met357 Thr located in the carboxyl terminus, which were transmitted from the patient' s father and mother, respectively, thereby giving rise to a compound heterozygote mutation in the patient.
The nonsense mutation, Arg338Stop, deletes the terminal 53 residues of the carboxyl tail that could alter phosphorylation at the tyrosine kinase site at Tyr337, and cause loss of function in the channel protein (6).
M357T
protein
substitution
true positive
P48048
We have identified amino acid exchanges Arg338Stop and Met357Thr in the gene exon 5 for ROMK by PCR and direct sequencing.
(A) Corresponding DNA sequence (sense strand) showing the Arg338Stop mutation; (B) Corresponding DNA sequence (sense strand) showing the Met357Thr mutation.
The Arg338Stop and Met357Thr mutations located in the C-terminal intracellular part were transmitted from the father and mother of the patient, respectively, thereby giving rise to a compound heterozygote mutation in the patient.
Arg 338Stop and Met357Thr had been previously reported by International Collaborative Study Group for Bartter-like syndromes (6) and Simon et al.
The function of the missense mutation, Met357Thr, was suggested to be normal (11).
However, the functional analysis of the missense mutation, Met357Thr, was done in vitro, and the effect due to regulation by ATP and pH was not completely eliminated.
Furthermore, her significant perinatal problems strongly suggest that the function of Met357Thr can be abnormal in vivo, and regulated by some factors in vitro.
T1631C
protein
substitution
true negative
The second mutation (T1631C) caused an exchange of Met357 for Thr (Fig.
12376531
full text
Y14124A
protein
substitution
true negative
Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity.
Typo
I1427A
protein
substitution
true positive
P13569
Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation.
Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis.
Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties.
Because the chloride channel activity and relative surface expression of Y1424A and I1427A CFTR are elevated to a similar extent, we propose that these substitutions affect protein trafficking but not CFTR chloride channel function.
For construction of the Y1424A,I1427A mutant, a BstXI-SgrAI fragment that coded for the COOH-terminal tail region of Y1424A CFTR was subcloned into pSK-Bluescript (Stratagene).
The levels of expression of wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were analyzed in COS-7 cells 48 h after transfection.
The relative amounts of wildtype (lanes 2 and 6), Y1424A (lanes 3 and 7), and Y1424A,I1427A CFTR (lanes 4 and 8) are shown.
COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR were surface-biotinylated and lysed in RIPA buffer (see "Materials and Methods").
The percentage CFTR at the cell surface was markedly increased for Y1424A,I1427A CFTR 0ompared with both wild-type (108% increase, n c 10, p .001) and Y1424A CFTR (59% increase, n 10, p 0.001) (Fig.
Mutations at Tyr1424 and Ile1427 Do Not Alter CFTR Maturation Efficiency or Protein Half-life--To test the effects of these mutations on maturation efficiency and protein half-life, we performed metabolic pulse-chase experiments on COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR.
2 show that the half-lives for wild-type (Wt), 2 1424A, and Y1424A,I1427A CFTR were 10.3 Y 2.3, 11.3 .6, and 11.3 1.5 h (mean S.D.).
The average maturation efficiency for wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were 32, 31, and 31%, respectively (bottom right panel).
This finding demonstrated that elevated surface expression of Y1424A,I1427A CFTR was not because of alterations in maturation efficiency.
COS-7 cells transfected with wild-type, Y1424A, or Y1424A,I1427A CFTR were analyzed 48-h post-transfection.
The percentage of wild-type, Y1424A, and Y1424A,I1427A CFTR internalized after 2.5 min was 34, 18, and 8 respectively.
CFTR Internalization Signals 49955 TABLE I Summary of whole cell patch clamp recordings for wild-type CFTR and for CFTR mutants shows elevated activity in the mutants relative to wild type cAMP-activated chloride currenta Transient transfection Non-green Control Wild type Green Y1424A pA at 100 mV Y1424A/I1427A Set 1 (Fold-difference) Set 2 (Fold-difference) Set 3 (Fold-difference) Fold-difference average 255 201 393 36b (13)c 68 (3) 140 (4) 95 (5) 1.0 738 52 (5) 1.0 1193 55 (7) 1.0 1.0 1070 1650 200* (5) 1.54 986 24* (5) 1.34 1767 164* (7) 1.48 1.45 0.06* 2125 195 (5) 1.99 1451 35 (5) 1.97 3424 205 (6) 2.87 2.28 0.30 a Three sets of transiently transfected COS-7 cells were analyzed in parallel with the protein biochemistry.
attributed to alterations in the internalization rate of CFTR, we performed internalization assays on COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR.
For Y1424A and Y1424A,I1427A CFTR, internalization dropped to 21 and 8%, respectively, during the same time period.
The Y1424A and Y1424A,I1427A CFTR Have Normal Chloride Channel Properties--Because the biochemical data suggested that a specific motif in the CFTR COOH terminus dramatically affected endocytosis and because point mutations in the NH2 terminus lead to both disruption of binding to docking machinery and changes in CFTR ion channel function, we tested whether the mutation of Tyr1424 and Ile1427 affected chloride channel function.
In agreement with the surface biotinylation assays, CFTR whole cell Cl currents in Y1424A CFTR and Y1424A,I1427A CFTRtransfected cells were elevated compared with wild-type CFTRc expressing cells (Table I), suggesting that the elevated Cl hannel activity was the result of the elevated surface expression of CFTR.
4, C and D, show the Y1424A and Y1424A,I1427A Cl currentvoltage relationships, respectively, and indicate that although the sensitivities to DIDS and glibenclamide remain similar to wild-type (Fig.
Single channel biophysical properties of wild-type, Y1424A, and Y1424A,I1427A CFTR were also assessed.
Representative recordings of wildtype, Y1424A, and Y1424A,I1427A CFTR at 50 60 mV (negative to pipette potential) are shown in Fig.
Nevertheless, the whole cell and single channel recordings together show that the difference in Cl channel activity is attributed to elevated surface expression without a significant change in CFTR chloride channel properties among wild-type, Y1424A, and Y1424A,I1427A CFTR.
In examining the mechanism for the elevated surface expression of CFTR, we first showed that total expression levels of wild-type, Y1424A, and Y1424A,I1427A were the same.
Typical whole-cell IV plots for wild-type CFTR (panel B), Y1424A (panel C), and Y1424A,I1427A (panel D) showing cyclic AMP-stimulated chloride currents in the absence of blockers (squares), presence of DIDS (upward triangles), and presence of glibenclamide (inverted triangles).
Moreover, we showed that Y1424A,I1427A CFTR was internalized much more slowly than the native protein (76% inhibition at 2.5 min) with an internalization rate of 2%/min.
Second, the internalization rate of Y1424A,I1427A CFTR is comparable with the rate of bulk flow lipid uptake via the endocytic pathway ( 2%/min.) (18), suggesting that the residual internalization activity observed in these studies reflects nonspecific uptake through clathrin-coated pits.
Y1424A
protein
substitution
true positive
P13569
Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L.
Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation.
Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis.
Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties.
We find that the substitution of Tyr1424 and Ile1427 with alanine residues resulted in a 2-fold increase in surface expression, whereas the single Y1424A mutation shows an intermediate phenotype.
Because the chloride channel activity and relative surface expression of Y1424A and I1427A CFTR are elevated to a similar extent, we propose that these substitutions affect protein trafficking but not CFTR chloride channel function.
The construction of the Y1424A mutant was described previously (3).
For construction of the Y1424A,I1427A mutant, a BstXI-SgrAI fragment that coded for the COOH-terminal tail region of Y1424A CFTR was subcloned into pSK-Bluescript (Stratagene).
The levels of expression of wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were analyzed in COS-7 cells 48 h after transfection.
The relative amounts of wildtype (lanes 2 and 6), Y1424A (lanes 3 and 7), and Y1424A,I1427A CFTR (lanes 4 and 8) are shown.
COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR were surface-biotinylated and lysed in RIPA buffer (see "Materials and Methods").
The percentage CFTR at the cell surface was markedly increased for Y1424A,I1427A CFTR 0ompared with both wild-type (108% increase, n c 10, p .001) and Y1424A CFTR (59% increase, n 10, p 0.001) (Fig.
Mutations at Tyr1424 and Ile1427 Do Not Alter CFTR Maturation Efficiency or Protein Half-life--To test the effects of these mutations on maturation efficiency and protein half-life, we performed metabolic pulse-chase experiments on COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR.
2 show that the half-lives for wild-type (Wt), 2 1424A, and Y1424A,I1427A CFTR were 10.3 Y 2.3, 11.3 .6, and 11.3 1.5 h (mean S.D.).
The average maturation efficiency for wild-type (Wt), Y1424A, and Y1424A,I1427A CFTR were 32, 31, and 31%, respectively (bottom right panel).
This finding demonstrated that elevated surface expression of Y1424A,I1427A CFTR was not because of alterations in maturation efficiency.
COS-7 cells transfected with wild-type, Y1424A, or Y1424A,I1427A CFTR were analyzed 48-h post-transfection.
The percentage of wild-type, Y1424A, and Y1424A,I1427A CFTR internalized after 2.5 min was 34, 18, and 8 respectively.
CFTR Internalization Signals 49955 TABLE I Summary of whole cell patch clamp recordings for wild-type CFTR and for CFTR mutants shows elevated activity in the mutants relative to wild type cAMP-activated chloride currenta Transient transfection Non-green Control Wild type Green Y1424A pA at 100 mV Y1424A/I1427A Set 1 (Fold-difference) Set 2 (Fold-difference) Set 3 (Fold-difference) Fold-difference average 255 201 393 36b (13)c 68 (3) 140 (4) 95 (5) 1.0 738 52 (5) 1.0 1193 55 (7) 1.0 1.0 1070 1650 200* (5) 1.54 986 24* (5) 1.34 1767 164* (7) 1.48 1.45 0.06* 2125 195 (5) 1.99 1451 35 (5) 1.97 3424 205 (6) 2.87 2.28 0.30 a Three sets of transiently transfected COS-7 cells were analyzed in parallel with the protein biochemistry.
attributed to alterations in the internalization rate of CFTR, we performed internalization assays on COS-7 cells expressing wild-type, Y1424A, and Y1424A,I1427A CFTR.
For Y1424A and Y1424A,I1427A CFTR, internalization dropped to 21 and 8%, respectively, during the same time period.
The Y1424A and Y1424A,I1427A CFTR Have Normal Chloride Channel Properties--Because the biochemical data suggested that a specific motif in the CFTR COOH terminus dramatically affected endocytosis and because point mutations in the NH2 terminus lead to both disruption of binding to docking machinery and changes in CFTR ion channel function, we tested whether the mutation of Tyr1424 and Ile1427 affected chloride channel function.
In agreement with the surface biotinylation assays, CFTR whole cell Cl currents in Y1424A CFTR and Y1424A,I1427A CFTRtransfected cells were elevated compared with wild-type CFTRc expressing cells (Table I), suggesting that the elevated Cl hannel activity was the result of the elevated surface expression of CFTR.
4, C and D, show the Y1424A and Y1424A,I1427A Cl currentvoltage relationships, respectively, and indicate that although the sensitivities to DIDS and glibenclamide remain similar to wild-type (Fig.
Single channel biophysical properties of wild-type, Y1424A, and Y1424A,I1427A CFTR were also assessed.
Representative recordings of wildtype, Y1424A, and Y1424A,I1427A CFTR at 50 60 mV (negative to pipette potential) are shown in Fig.
Nevertheless, the whole cell and single channel recordings together show that the difference in Cl channel activity is attributed to elevated surface expression without a significant change in CFTR chloride channel properties among wild-type, Y1424A, and Y1424A,I1427A CFTR.
In examining the mechanism for the elevated surface expression of CFTR, we first showed that total expression levels of wild-type, Y1424A, and Y1424A,I1427A were the same.
Typical whole-cell IV plots for wild-type CFTR (panel B), Y1424A (panel C), and Y1424A,I1427A (panel D) showing cyclic AMP-stimulated chloride currents in the absence of blockers (squares), presence of DIDS (upward triangles), and presence of glibenclamide (inverted triangles).
Moreover, we showed that Y1424A,I1427A CFTR was internalized much more slowly than the native protein (76% inhibition at 2.5 min) with an internalization rate of 2%/min.
Second, the internalization rate of Y1424A,I1427A CFTR is comparable with the rate of bulk flow lipid uptake via the endocytic pathway ( 2%/min.) (18), suggesting that the residual internalization activity observed in these studies reflects nonspecific uptake through clathrin-coated pits.
12798629
full text
C6B
protein
substitution
true negative
We used an additional set of MAbs provided by the National Institute for Biological Standards and Control (NIBSC), Potters Barr, UK, for serotypes 1 (MN3C6B-95/680), 14 (MN5C8C-95/688) and 21 (6B11-F2-B5-95/692) and for serosubtypes P1.7 (MN14C11.6-95/706), P1.10 (MN20F4.17-95/710), P1.12 (MN20A7.10-95/712), P1.14 (MN21G3.17-95/716), P1.5 (MN22A9.19-95/702).
14663144
full text
D66N
protein
substitution
true negative
The primers used were: E47Q, sense 5 -AAA TCT TCC T TC ATC TGC A AC ACA-3 and antisense 5 -CTG ATC ACC CCA CAC ACT CTC-3 creating a new BclI site; D66N, sense 5 -TAT AAC CA A T TC T TC CCC ATC TC-3 and antisense 5 -GCA GAC GCT GT T GCA GCC AGG-3 , creating a new AlwNI site; D169N, sense 5 -AAC GTC TAC CCC TGC CCC A AC-3 and antisense 5 -GCA CT T GAC CAG CCG CAC CAT-3 , suppressing a AatII site; D178N, sense 5 -AAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , destroying a BstXI site; and D178Y, sense 5 -TAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , remov ing a BstXI site.
To identif y the Cx32 residues that interact w ith Ca2 , we introduced substitutions in the E1 loop (E47Q and D66N) and in the E2 domain (D169N and D178N), replacing carbox ylate-c ont ain ing residues w ith polar uncharged amino acids.
D178N
protein
substitution
true negative
The primers used were: E47Q, sense 5 -AAA TCT TCC T TC ATC TGC A AC ACA-3 and antisense 5 -CTG ATC ACC CCA CAC ACT CTC-3 creating a new BclI site; D66N, sense 5 -TAT AAC CA A T TC T TC CCC ATC TC-3 and antisense 5 -GCA GAC GCT GT T GCA GCC AGG-3 , creating a new AlwNI site; D169N, sense 5 -AAC GTC TAC CCC TGC CCC A AC-3 and antisense 5 -GCA CT T GAC CAG CCG CAC CAT-3 , suppressing a AatII site; D178N, sense 5 -AAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , destroying a BstXI site; and D178Y, sense 5 -TAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , remov ing a BstXI site.
To identif y the Cx32 residues that interact w ith Ca2 , we introduced substitutions in the E1 loop (E47Q and D66N) and in the E2 domain (D169N and D178N), replacing carbox ylate-c ont ain ing residues w ith polar uncharged amino acids.
A lthough none of these substitutions inter fered w ith the abilit y to make hemichannels, the D169N and the D178N hemichannels responded to volt age pulses by producing less variable amplitudes of macrosc opic currents than those obser ved in w ild-t ype hemichannels ex posed to changes in [Ca2 ]o (i.e., the changes at 0.5 mM Ca2 were not so large; Fig.
First, on depolarization, the D169N or D178N hemichannel o p e n e d t o b o t h 18 a n d 90 c o n d u c t a n c e s u b l e v e l s ; a n d s e c o n d , t h e f l i c k e r i n g i n t e r r u p t i o n s o f t h e 18 u n i t a r y c u r r e n t s d i s a p peared at negative volt ages (Fig.
Thus, in nor mal [Ca2 ]o, the D169N and D178N hemichannels behaved as w ild-t ype hemichannels in the absence of divalent cations.
To discern bet ween these t wo possibilities, equal amounts of RNA for the t wo single D169N and D178N mut ants were c oinjected into ooc y tes.
Interestingly, the for mation of heteromeric D169N and D178N hemichannels was able so rescue most of the Ca2 -dependent properties.
This rec over y suggests that the D169 residue of the D178N subun it and the D178 f rom the adjacent D169N subun it must be arranged in a precise way to allow the interaction w ith Ca2 to oc cur.
Further more, the oc cupanc y of three of these sites would be suf ficient to partially oc clude the lumen and block full volt age gating, because this is the max imal number of theoretical binding sites that c ould result f rom the c ombination of the t wo mut ant subun its in heteromeric D169N and D178N hemichannels (see Fig.
(A) Slow-activating outward currents of the single neutralizing D169N and D178N mutant hemichannels elicited by the voltage-pulse protocol of Fig.
Curves of homomeric mutant hemichannels (D169N, D178N, and D178Y) showed a large reduction in Ca2 sensitivity vs.
The formation of heteromeric hemichannels by coexpressing the two mutant D169N and D178N subunits reintroduced moderate Ca2 sensitivity; asterisks indicate significant differences relative to each mutant alone (t test, P 0.05).
(C) Single-channel recordings of homomeric mutant hemichannels (D169N, D178N, and D178Y) in a normal ND96 solution.
(D) Selected unitary recordings of the oocytes coinjected with D169N and D178N mutants in a normal ND96 solution.
D178Y
protein
substitution
true negative
Interestingly, a naturally occurring mutation (D178Y) that d uses an inherited peripheral neuropathy induces a complete Ca2 ca eregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.
The primers used were: E47Q, sense 5 -AAA TCT TCC T TC ATC TGC A AC ACA-3 and antisense 5 -CTG ATC ACC CCA CAC ACT CTC-3 creating a new BclI site; D66N, sense 5 -TAT AAC CA A T TC T TC CCC ATC TC-3 and antisense 5 -GCA GAC GCT GT T GCA GCC AGG-3 , creating a new AlwNI site; D169N, sense 5 -AAC GTC TAC CCC TGC CCC A AC-3 and antisense 5 -GCA CT T GAC CAG CCG CAC CAT-3 , suppressing a AatII site; D178N, sense 5 -AAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , destroying a BstXI site; and D178Y, sense 5 -TAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , remov ing a BstXI site.
Curves of homomeric mutant hemichannels (D169N, D178N, and D178Y) showed a large reduction in Ca2 sensitivity vs.
(C) Single-channel recordings of homomeric mutant hemichannels (D169N, D178N, and D178Y) in a normal ND96 solution.
In this c ontext, we show here that the D178Y mut ant that destroyed the divalent cation-binding site caused a c omplete loss of the blocking actions exerted by Ca2 on hemichannel activ it y (Fig.
D169N
protein
substitution
true negative
The primers used were: E47Q, sense 5 -AAA TCT TCC T TC ATC TGC A AC ACA-3 and antisense 5 -CTG ATC ACC CCA CAC ACT CTC-3 creating a new BclI site; D66N, sense 5 -TAT AAC CA A T TC T TC CCC ATC TC-3 and antisense 5 -GCA GAC GCT GT T GCA GCC AGG-3 , creating a new AlwNI site; D169N, sense 5 -AAC GTC TAC CCC TGC CCC A AC-3 and antisense 5 -GCA CT T GAC CAG CCG CAC CAT-3 , suppressing a AatII site; D178N, sense 5 -AAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , destroying a BstXI site; and D178Y, sense 5 -TAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , remov ing a BstXI site.
To identif y the Cx32 residues that interact w ith Ca2 , we introduced substitutions in the E1 loop (E47Q and D66N) and in the E2 domain (D169N and D178N), replacing carbox ylate-c ont ain ing residues w ith polar uncharged amino acids.
A lthough none of these substitutions inter fered w ith the abilit y to make hemichannels, the D169N and the D178N hemichannels responded to volt age pulses by producing less variable amplitudes of macrosc opic currents than those obser ved in w ild-t ype hemichannels ex posed to changes in [Ca2 ]o (i.e., the changes at 0.5 mM Ca2 were not so large; Fig.
First, on depolarization, the D169N or D178N hemichannel o p e n e d t o b o t h 18 a n d 90 c o n d u c t a n c e s u b l e v e l s ; a n d s e c o n d , t h e f l i c k e r i n g i n t e r r u p t i o n s o f t h e 18 u n i t a r y c u r r e n t s d i s a p peared at negative volt ages (Fig.
Thus, in nor mal [Ca2 ]o, the D169N and D178N hemichannels behaved as w ild-t ype hemichannels in the absence of divalent cations.
To discern bet ween these t wo possibilities, equal amounts of RNA for the t wo single D169N and D178N mut ants were c oinjected into ooc y tes.
Interestingly, the for mation of heteromeric D169N and D178N hemichannels was able so rescue most of the Ca2 -dependent properties.
This rec over y suggests that the D169 residue of the D178N subun it and the D178 f rom the adjacent D169N subun it must be arranged in a precise way to allow the interaction w ith Ca2 to oc cur.
Further more, the oc cupanc y of three of these sites would be suf ficient to partially oc clude the lumen and block full volt age gating, because this is the max imal number of theoretical binding sites that c ould result f rom the c ombination of the t wo mut ant subun its in heteromeric D169N and D178N hemichannels (see Fig.
(A) Slow-activating outward currents of the single neutralizing D169N and D178N mutant hemichannels elicited by the voltage-pulse protocol of Fig.
Curves of homomeric mutant hemichannels (D169N, D178N, and D178Y) showed a large reduction in Ca2 sensitivity vs.
The formation of heteromeric hemichannels by coexpressing the two mutant D169N and D178N subunits reintroduced moderate Ca2 sensitivity; asterisks indicate significant differences relative to each mutant alone (t test, P 0.05).
(C) Single-channel recordings of homomeric mutant hemichannels (D169N, D178N, and D178Y) in a normal ND96 solution.
(D) Selected unitary recordings of the oocytes coinjected with D169N and D178N mutants in a normal ND96 solution.
E47Q
protein
substitution
true negative
The primers used were: E47Q, sense 5 -AAA TCT TCC T TC ATC TGC A AC ACA-3 and antisense 5 -CTG ATC ACC CCA CAC ACT CTC-3 creating a new BclI site; D66N, sense 5 -TAT AAC CA A T TC T TC CCC ATC TC-3 and antisense 5 -GCA GAC GCT GT T GCA GCC AGG-3 , creating a new AlwNI site; D169N, sense 5 -AAC GTC TAC CCC TGC CCC A AC-3 and antisense 5 -GCA CT T GAC CAG CCG CAC CAT-3 , suppressing a AatII site; D178N, sense 5 -AAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , destroying a BstXI site; and D178Y, sense 5 -TAC TGC T TC GTG TCC CGC C-3 and antisense 5 -CAC TGT GT T GGG GCA GGG GT-3 , remov ing a BstXI site.
To identif y the Cx32 residues that interact w ith Ca2 , we introduced substitutions in the E1 loop (E47Q and D66N) and in the E2 domain (D169N and D178N), replacing carbox ylate-c ont ain ing residues w ith polar uncharged amino acids.
12930913
full text
12138168
full text
M1305Q
protein
substitution
P15390
true positive
This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/ I1303Q/F1304Q/ F1304Q/M1305Q.
In DIV-A1529D M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the 1 subunit.
1) and will subsequently be referred to as "ultraslow fraction." In the case of the mutant 1-I1303Q/F1304Q/M1305Q, which did not exhibit ultra-slow inactivation, recovery from slow inactivation was fit with the monoexponential function.
Mutagenesis of DIV-A1529D I1303Q/F1304Q/M1305Q--I1303Q/ F1304Q/M1305Q was made by oligonucleotide-directed mutagenesis and confirmed by sequencing the polymerized region.
DIV-A1529D was introduced in this construct by cloning the SacII-KpnI fragment into the I1303Q/F1304Q/M1305Q construct.
Effects of Destabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels The Inactivation-defective DIV-A1529D Channel Mutant I1303Q/F1304Q/M1305Q Enhances Ultra-slow Inactivation-- The strategy of all the experiments presented above was based on the stabilization of the fast inactivated state by different methods.
9 shows the growth of inward currents through inactivationdefective DIV-A1529D I1303Q/F1304Q/M1305Q channels with subsequent pulses at 20-s intervals.
Bi-exponential curve fits to the data points (solid lines, see "Experimental Procedures") were used to estimate the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that had entered the ultra-slow inactivated state after a 300-s prepulse to 50 and 20 mV.
Comparing these fractions between inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels and DIV-A1529D channels, it can be noticed that a prepulse potential of 50 mV produced similar fractions of channels recovering from ultra-slow inactivation in both cases, whereas a prepulse potential of 20 mV produced considerable ultra-slow inactivation only in inactivation-defective channels.
9C is the time course of recovery from a 300-s inactivating prepulse to 20 mV in I1303Q/F1304Q/M1305Q channels that did not carry the additional DIV-A1529D mutation in the selectivity filter (n 7).
The fact that adding the "selectivity filter mutation" DIV-A1529D to the "inactivation gate mutation" I1303Q/ F1304Q/M1305Q changes the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/ I M1305Q to a bi-exponential time course in DIV-A1529D 1303Q/F1304Q/M1305Q suggests that the mutation DIVA1529D created an additional inactivated state, i.e.
Stabilization of the fast inactivated state by a strongly depolarized potential ( 20 mV) dramatically inhibits entry to the ultra-slow inactivated state in DIV-A1529D channels but not in inactivation-defective DIV-A1529D I1303Q/F1304Q/ M1305Q channels (Fig.
In the latter, the fast inactivated state cannot be efficiently stabilized due to the mutations I1303Q/F1304Q/M1305Q.
If the fast inactivated state cannot be stabilized in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels, coexpression of the 1 subunit, which stabilizes the fast inactivated state in DIV-A1529D channels should not affect the fraction of DIV-A1529D I1303Q/F1304Q/M1305Q channels 37112 Sodium Channel Inactivation Gate and Channel Structure FIG.
Ultra-slow inactivation in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
A, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q.
For comparison, recovery from 300-s prepulses to 20 mV in the single mutant 1-I1303Q/ F1304Q/M1305Q is shown (n 7).
Whereas the time course of recovery in DIV-A1529D I1303Q/F1304Q/M1305Q (DIV-ADQQQ) was best fit by a bi-exponential equation (Equation 1), the time course of recovery in 1- I1303Q/F1304Q/M1305Q was best fit by a mono-exponential function (Equation 2).
D, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q 1 channels ( / 1 ratio, 0.3).
Recovery from ultra-slow inactivation was similar to DIV-A1529D I1303Q/F1304Q/M1305Q -only channels (compare with B).
9D I shows the growth of inward currents through DIV-A1529D 1303Q/F1304Q/M1305Q 1 channels with subsequent pulses at 20-s intervals.
Hence, coexpression of the 1 subunit with DIV-A1529D 1303Q/F1304Q/M1305Q channels did not affect ultra-slow inactivation.
To destabilize the fast inactivated state, we used DIV-A1529D channels with the additional mutations I1303Q/F1304Q/M1305Q, which selectively remove fast inactivation (3, 4, 32).
Indeed, a considerable fraction of fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels could be driven to the ultra-slow inactivated state during a 300-s depolarization to 20 mV (Fig.
Nevertheless, the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that entered the ultra-slow inactivated state after a 300-s prepulse to 20 mV, 0.44, was significantly smaller than the corresponding fraction after a 300-s prepulse to 50 mV, 0.58.
This indicated that stronger depolarizations modestly inhibited entry to the ultra-slow inactivated state also in fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
The fact that the fast inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels still exhibited a "residual" U-shaped voltage dependence of ultra-slow inactivation supports the notion that ultra-slow inactivation in this mutant is reached via transitions from pre-open states.
Whereas coexpression of the 1 subunit significantly reduced ultra-slow inactivation in DIV-A1529D channels, ultra-slow inactivation was not affected by 1 in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels (Fig.
Accordingly, in our experiments we observed an accelerated current decay in DIV-A1529D I1303Q/ F1304Q/M1305Q 1 channels (compare current traces of Fig.
Selective removal of fast inactivation by the mutation I1303Q/F1304Q/M1305Q allows slow inactivation to occur more quickly and completely (17, 33).
First, the addition of the selectivity filter mutation DIVA1529D to the inactivation gate mutation I1303Q/F1304Q/ M1305Q changed the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/M1305Q to a biexponential time course in DIV-A1529D I1303Q/F1304Q/ M1305Q (Fig.
R13Q
protein
substitution
true negative
The reduction of entry to the ultra-slow inactivated state by the mutant -CTX R13Q led us to propose that ultra-slow inactivation most likely reflects a rearrangement of the outer pore region and that binding of the mutant -CTX to the outer pore stabilizes the FIG.
I1303Q
protein
substitution
P15390
true positive
This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/ I1303Q/F1304Q/ F1304Q/M1305Q.
1) and will subsequently be referred to as "ultraslow fraction." In the case of the mutant 1-I1303Q/F1304Q/M1305Q, which did not exhibit ultra-slow inactivation, recovery from slow inactivation was fit with the monoexponential function.
Mutagenesis of DIV-A1529D I1303Q/F1304Q/M1305Q--I1303Q/ F1304Q/M1305Q was made by oligonucleotide-directed mutagenesis and confirmed by sequencing the polymerized region.
DIV-A1529D was introduced in this construct by cloning the SacII-KpnI fragment into the I1303Q/F1304Q/M1305Q construct.
Effects of Destabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels The Inactivation-defective DIV-A1529D Channel Mutant I1303Q/F1304Q/M1305Q Enhances Ultra-slow Inactivation-- The strategy of all the experiments presented above was based on the stabilization of the fast inactivated state by different methods.
9 shows the growth of inward currents through inactivationdefective DIV-A1529D I1303Q/F1304Q/M1305Q channels with subsequent pulses at 20-s intervals.
Bi-exponential curve fits to the data points (solid lines, see "Experimental Procedures") were used to estimate the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that had entered the ultra-slow inactivated state after a 300-s prepulse to 50 and 20 mV.
Comparing these fractions between inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels and DIV-A1529D channels, it can be noticed that a prepulse potential of 50 mV produced similar fractions of channels recovering from ultra-slow inactivation in both cases, whereas a prepulse potential of 20 mV produced considerable ultra-slow inactivation only in inactivation-defective channels.
9C is the time course of recovery from a 300-s inactivating prepulse to 20 mV in I1303Q/F1304Q/M1305Q channels that did not carry the additional DIV-A1529D mutation in the selectivity filter (n 7).
The fact that adding the "selectivity filter mutation" DIV-A1529D to the "inactivation gate mutation" I1303Q/ F1304Q/M1305Q changes the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/ I M1305Q to a bi-exponential time course in DIV-A1529D 1303Q/F1304Q/M1305Q suggests that the mutation DIVA1529D created an additional inactivated state, i.e.
Stabilization of the fast inactivated state by a strongly depolarized potential ( 20 mV) dramatically inhibits entry to the ultra-slow inactivated state in DIV-A1529D channels but not in inactivation-defective DIV-A1529D I1303Q/F1304Q/ M1305Q channels (Fig.
In the latter, the fast inactivated state cannot be efficiently stabilized due to the mutations I1303Q/F1304Q/M1305Q.
If the fast inactivated state cannot be stabilized in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels, coexpression of the 1 subunit, which stabilizes the fast inactivated state in DIV-A1529D channels should not affect the fraction of DIV-A1529D I1303Q/F1304Q/M1305Q channels 37112 Sodium Channel Inactivation Gate and Channel Structure FIG.
Ultra-slow inactivation in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
A, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q.
For comparison, recovery from 300-s prepulses to 20 mV in the single mutant 1-I1303Q/ F1304Q/M1305Q is shown (n 7).
Whereas the time course of recovery in DIV-A1529D I1303Q/F1304Q/M1305Q (DIV-ADQQQ) was best fit by a bi-exponential equation (Equation 1), the time course of recovery in 1- I1303Q/F1304Q/M1305Q was best fit by a mono-exponential function (Equation 2).
D, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q 1 channels ( / 1 ratio, 0.3).
Recovery from ultra-slow inactivation was similar to DIV-A1529D I1303Q/F1304Q/M1305Q -only channels (compare with B).
To destabilize the fast inactivated state, we used DIV-A1529D channels with the additional mutations I1303Q/F1304Q/M1305Q, which selectively remove fast inactivation (3, 4, 32).
Indeed, a considerable fraction of fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels could be driven to the ultra-slow inactivated state during a 300-s depolarization to 20 mV (Fig.
Nevertheless, the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that entered the ultra-slow inactivated state after a 300-s prepulse to 20 mV, 0.44, was significantly smaller than the corresponding fraction after a 300-s prepulse to 50 mV, 0.58.
This indicated that stronger depolarizations modestly inhibited entry to the ultra-slow inactivated state also in fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
The fact that the fast inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels still exhibited a "residual" U-shaped voltage dependence of ultra-slow inactivation supports the notion that ultra-slow inactivation in this mutant is reached via transitions from pre-open states.
Whereas coexpression of the 1 subunit significantly reduced ultra-slow inactivation in DIV-A1529D channels, ultra-slow inactivation was not affected by 1 in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels (Fig.
Accordingly, in our experiments we observed an accelerated current decay in DIV-A1529D I1303Q/ F1304Q/M1305Q 1 channels (compare current traces of Fig.
Selective removal of fast inactivation by the mutation I1303Q/F1304Q/M1305Q allows slow inactivation to occur more quickly and completely (17, 33).
First, the addition of the selectivity filter mutation DIVA1529D to the inactivation gate mutation I1303Q/F1304Q/ M1305Q changed the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/M1305Q to a biexponential time course in DIV-A1529D I1303Q/F1304Q/ M1305Q (Fig.
A1529D
protein
substitution
P15390
true positive
Dudley , and Hannes Todt** From the Institute of Pharmacology, University of Vienna, A-1090 Vienna, Austria, the Cardiac Electrophysiology Laboratories, The University of Chicago, Chicago, Illinois 60637, and the Division of Cardiology, Emory University, Atlanta, Georgia 30033 and the Atlanta Veterans Affairs Hospital, Decatur, Georgia 30033 Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na hannel 1 (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of 100 s.
In DIV-A1529D M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the 1 subunit.
These results strongly suggest that FI inhibits USI in DIV-A1529D channels.
We recently demonstrated that replacement of alanine 1529 by aspartic acid in the DIV P-loop of the rat skeletal muscle Na channel 1 (DIV-A1529D) enhanced entry to an ultra-slow inactivated state, which is characterized by time constants of entry to and recovery from inactivation of 100 s (1).
In DIV-A1529D channels, transition to this ultra-slow inactivated state was substantially reduced by binding to the outer pore of a mutant -CTX.1 This suggested that ultra-slow inactivation may reflect a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide can stabilize the pore structure (1, 16).
In a previous study (1), we found that coexpression of the rat brain 1 subunit slowed entry of DIV-A1529D channels to the ultra-slow inactivated state during long-lasting depolarizations.
Mutagenesis of the 1--The oligonucleotide-directed point mutation DIV-A1529D was introduced using four-primer PCR.
Mutagenesis of DIV-A1529D I1303Q/F1304Q/M1305Q--I1303Q/ F1304Q/M1305Q was made by oligonucleotide-directed mutagenesis and confirmed by sequencing the polymerized region.
DIV-A1529D was introduced in this construct by cloning the SacII-KpnI fragment into the I1303Q/F1304Q/M1305Q construct.
In the case of DIV-A1529D channels, the injected molar cRNA / 1 ratio ranged from 100 to 0.07.
The kinetics of the current decay after activation by a step depolarization was taken as a measure of the fraction of DIV-A1529D channels, which inactivated fast (time constants about 1 ms ("fast-gating channels") or slow (time constants 10 ms ("slow-gating channels")).
For accurate comparison of the time constants of current decay in DIV-A1529D -only and DIV-A1529D 1 channels, it was essential to precisely adjust the bath temperature to 20 C (see above).
RESULTS Recovery from Ultra-slow Inactivation in 1 and DIV-A1529D Channels Fig.
Ultra-slow inactivation in 1 and DIV-A1529D channels.
Growth of inward current during recovery from ultra-slow inactivation in wild type 1 (A) and mutant DIV-A1529D (B) Na channels expressed in Xenopus laevis oocytes.
Recovery from ultra-slow inactivation in mutant DIV-A1529D took considerably longer than in 1 channels.
Typical examples of original traces of inward currents through wild type 1 and 1 1 (A) channels or mutant DIVA1529D and DIV-A1529D 1 (B) channels elicited by test pulses from 120 to 20 mV.
Both in 1 and mutant DIV-A1529D channels, coexpression of the 1 subunit dramatically accelerated the current decay.
In contrast to 1 channels, mutant DIV-A1529D exhibited a large ultra-slow recovering component of inactivation (Fig.
In a series of such experiments, the calculated fractions of 0 channels recovering from ultra-slow inactivation were 0.22 .02 (n 6) in 1 channels and 0.60 0.02 (n 19) in DIV-A1529D (value for 1 taken from Hilber et al.
Thus, significantly more channels recovered from ultra-slow inactivation in DIV-A1529D than in 1 channels.
A more detailed investigation of ultra-slow inactivation in DIV-A1529D channels was carried out in a previous study (1).
Effects of Stabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels To investigate the relationship between fast inactivation and ultra-slow inactivation, we used different strategies to stabilize the fast inactivated state and tested the effects of these strategies on ultra-slow inactivation.
Here, we used coexpression of the 1 subunit to stabilize the fast inactivated state in DIV-A1529D channels to investigate the effects of fast inactivation on ultra-slow inactivation.
First, we confirmed that the 1 subunit exhibited similar modulatory effects in DIV-A1529D channels as previously reported for wild type Na channels (22, 23, 31).
We compared the current decay in oocytes that were injected either with DIV-A1529D -only cRNA or with DIV-A1529D rat brain c 1 cRNA (molar / 1 ratios 0.30).
A similar result was obtained with DIV-A1529D -only and 1 channels (Fig.
A bi-exponential funcDIV-A1529D tion (Equation 1) was fit to the decay currents, and the fractions of channels that inactivated with a fast and a slow time constant were calculated (see "Experimental Procedures").
The table shows that both 1 and 2 were similar in DIV-A1529D -only and DIV-A1529D 1 channels.
In DIV-A1529D -only injected oocytes only a small fraction of channels inactivated with a fast time constant.
DIV-A1529D 1 channels mainly inactivated with a fast time constant, but in all oocytes investigated, a small but discernable fraction of channels, about 0.07, inactivated with a slow time constant (Table I).
These results show that, as in wild type 1 channels (22, 23), coexpression of the 1 subunit dramatically increased the fraction of fast-gating DIV-A1529D channels, whereas the time constants themselves were not altered.
Thus, coexpression of 37108 Sodium Channel Inactivation Gate and Channel Structure TABLE I Parameters of bi-exponential current decay fits Equation 1) of DIV-A1529D -only and DIV-A1529D 1 channels expressed in Xenopus oocytes The channels were activated from a holding potential of 120 mV with the voltage step, which elicited maximum current ( 20 mV).
DIV-A1529D 1 (ms) 2 (ms) Fast fraction (A1) -only 1 Mean S.E.
the 1 subunit exhibits similar effects on the gating properties of DIV-A1529D and 1 channels, stabilizing the fast inactivated state in DIV-A1529D channels as well.
Coexpression of the 1 Subunit Delays Entry to the Ultra-slow Inactivated State in DIV-A1529D Channels--In a previous study (1) we investigated the effects of coexpression of the rat brain 1 subunit on ultra-slow inactivation in DIV-A1529D channels.
3 shows the recovery of inward currents through DIVA1529D 1 channels with subsequent pulses at 20-s intervals after being inactivated by a 300-s (A) or a 1200-s (B) depolarizing prepulse to 50 mV.
A summary of the tivation in DIV-A1529D recovery curves of four such experiments is shown in the inset.
Bi-exponential curve fits to the data points (solid lines) were used to estimate the fraction of DIV-A1529D 1 channels, which had entered the ultra-slow inactivated state (see "Experimental Procedures").
In DIV-A1529D -only channels, the corresponding fraction after a 300-s prepulse to 50 mV was 0.60 0 .02 (n 19); this fraction was not further increased when longer prepulse durations were used (see Ref.
These data confirm that the 1 subunit prolongs entry to the ultra-slow inactivated state in DIV-A1529D channels.
The 1 Subunit Effect on Ultra-slow Inactivation in DIVA1529D Channels Depends on the Molar cRNA / 1 Ratio--To explore the relationship between the 1-induced stabilization of the fast inactivated state and ultra-slow inactivation in more detail, we compared the amount of ultra-slow inactivation produced by a 300-s prepulse to 50 mV with the fraction of channels that showed a fast current decay after activation (the fast-gating fraction).
Xenopus oocytes were injected either with DIV-A1529D -only cRNA or with both DIV-A1529D and 1 subunit cRNA.
Coexpression of the 1 subunit in DIV-A1529D and ultra-slow inactivation.
Growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D 1 channels after a 300-s (A) or 1200-s (B) depolarizing prepulse to 50 mV (experimental design identical to Fig.
4A shows a linear correlation between the fraction of DIV-A1529D channels exhibiting a fast current decay and the fraction of channels that recovered from ultra-slow inactivation; the larger the fast-gating channel fraction, the smaller was the channel fraction recovering from ultra-slow inactivation.
4B shows a linear correlation between the fraction of fast-gating DIVA1529D channels (reflected by small current-time integral values) and the fraction of channels that recovered from ultra-slow inactivation; also here, the larger the fraction of fast-gating channels, the smaller was the channel fraction recovering from ultra-slow inactivation.
Current decay and ultra-slow inactivation in DIVA1529D 1 channels.
Comparison between the fraction of DIV-A1529D channels (empty bars) recovering from ultra-slow inactivation after a 100-s pulse train, during which repetitive 2-ms step depolarizations to 20 mV were followed by 28-ms periods at 120 mV (30 2-ms train protocol), and after a 100-s pulse train, during which 28-ms step depolarizations to 20 mV were followed by 2-ms periods at 120 mV.
In DIV-A1529D 1 channels (hatched bars) (molar ratio of / 1 subunit cRNA 0.3) neither the 30 2- nor the 30 28-ms train protocol elicited considerable ultra-slow inactivation.
The 1 Subunit Reduces Ultra-slow Inactivation Produced by Trains of Brief Depolarizations in DIV-A1529D Channels-- Until now, we have described the effects of 1 coexpression on ultra-slow inactivation produced by prolonged continuous depolarizations.
In a previous study (1), we showed that ultraslow inactivation could also be produced by brief repetitive depolarizations that enhanced the probability of channels to undergo transitions between closed and open states in DIVA1529D channels.
5 presents a comparison between the fractions of DIVA1529D channels (n 8) recovering from ultra-slow inactivation after a 100-s pulse train in which repetitive 2-ms step depolarizations to 20 mV were followed by 28-ms periods at 120 mV (30 2-ms train protocol) and after a 100-s pulse train in which 28-ms step depolarizations to 20 mV were followed by 2-ms interpulse intervals at 120 mV (30 28-ms train protocol).
These results suggest that functional association of the 1 subunit with DIV-A1529D subunits prevents ultra-slow inactivation produced by pulse train protocols.
2B indeed 1 suggests that a considerable fraction of DIV-A1529D channels entered the fast inactivated state during a 2-ms de- 37110 Sodium Channel Inactivation Gate and Channel Structure polarization, whereas in DIV-A1529D -only channels, this was not the case.
Naturally Occurring Fast Current Decay Reduces Ultra-slow Inactivation in DIV-A1529D -Only Channels--The observed kinetic effects of 1-coexpression are most likely the result of a 1-induced shift of equilibria between slow-mode-gating and fast-mode-gating channels.
To investigate whether the reduction of ultra-slow inactivation due to the coexpression of the 1 subunit was caused by stabilization of the "naturally occurring" fast inactivated state as opposed to the induction of a new kinetic state, we compared ultra-slow inactivation in DIV-A1529D -onlyinjected oocytes, which naturally exhibited different fast-gating channel fractions.
In general, currents of DIV-A1529D -only-injected oocytes had current time integrals 10, which is in excellent agreement with reported data from wild type -only channels (25).
Low Holding Potentials Favor Fast Current Decay and Reduce Ultra-slow Inactivation in DIV-A1529D -Only Channels--To further explore whether in the absence of the 1 subunit stabilization of fast inactivation inhibits entry to the ultra-slow inactivated state, we investigated if a slow-to-fast shift in gating forced by a low holding potential (23) also reduces ultra-slow inactivation in DIV-A1529D -only channels.
Therefore, we compared recovery from ultra-slow inactivation in single DIV-A1529D -only-injected oocytes at two different holding potentials, 120 and 80 mV.
7 shows the growth of inward current through DIV-A1529D -only channels with subsequent pulses at 20-s intervals after the channels were first inactivated by a 300-s depolarizing prepulse to 50 mV.
These data further confirm that the reduction of ultra-slow inactivation by coexpression of the 1 subunit in DIV-A1529D channels is caused by a shift from slow to fast channel gating.
Current decay and ultra-slow inactivation in DIVA1529D -only channels.
DIV-A1529D channels were pooled into two groups on the basis of current-time integrals (see Fig.
DIV-A1529D -only channels with current-time integrals 10, i.e.
Growth of inward current during recovery from ultra-slow inactivation in mutant DIV-A1529D channels at 120 mV (A) and 80 mV (B).
that the voltage dependence of ultra-slow inactivation was U-shaped with a local maximum at about 60 mV in DIVA1529D channels (1).
Here, we compared the relationship between the voltage dependence of ultra-slow inactivation and steady-state fast inactivation in DIV-A1529D channels.
8 shows the voltage dependence of ultra-slow inactivation and steady state fast inactivation in DIV-A1529D channels.
These experiments strongly suggest that stabilization of the fast inactivated state directly inhibits the transition to the ultra-slow inactivated state in DIV-A1529D channels.
Effects of Destabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels The Inactivation-defective DIV-A1529D Channel Mutant I1303Q/F1304Q/M1305Q Enhances Ultra-slow Inactivation-- The strategy of all the experiments presented above was based on the stabilization of the fast inactivated state by different methods.
9 shows the growth of inward currents through inactivationdefective DIV-A1529D I1303Q/F1304Q/M1305Q channels with subsequent pulses at 20-s intervals.
Bi-exponential curve fits to the data points (solid lines, see "Experimental Procedures") were used to estimate the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that had entered the ultra-slow inactivated state after a 300-s prepulse to 50 and 20 mV.
In DIV-A1529D channels that were not inactivation-defective, the corresponding fractions were 0.60 0.02 (n 19) and 0.13 0.02 (n 7).
Comparing these fractions between inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels and DIV-A1529D channels, it can be noticed that a prepulse potential of 50 mV produced similar fractions of channels recovering from ultra-slow inactivation in both cases, whereas a prepulse potential of 20 mV produced considerable ultra-slow inactivation only in inactivation-defective channels.
9C is the time course of recovery from a 300-s inactivating prepulse to 20 mV in I1303Q/F1304Q/M1305Q channels that did not carry the additional DIV-A1529D mutation in the selectivity filter (n 7).
The fact that adding the "selectivity filter mutation" DIV-A1529D to the "inactivation gate mutation" I1303Q/ F1304Q/M1305Q changes the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/ I M1305Q to a bi-exponential time course in DIV-A1529D 1303Q/F1304Q/M1305Q suggests that the mutation DIVA1529D created an additional inactivated state, i.e.
Stabilization of the fast inactivated state by a strongly depolarized potential ( 20 mV) dramatically inhibits entry to the ultra-slow inactivated state in DIV-A1529D channels but not in inactivation-defective DIV-A1529D I1303Q/F1304Q/ M1305Q channels (Fig.
If the fast inactivated state cannot be stabilized in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels, coexpression of the 1 subunit, which stabilizes the fast inactivated state in DIV-A1529D channels should not affect the fraction of DIV-A1529D I1303Q/F1304Q/M1305Q channels 37112 Sodium Channel Inactivation Gate and Channel Structure FIG.
Ultra-slow inactivation in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
A, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q.
Whereas the time course of recovery in DIV-A1529D I1303Q/F1304Q/M1305Q (DIV-ADQQQ) was best fit by a bi-exponential equation (Equation 1), the time course of recovery in 1- I1303Q/F1304Q/M1305Q was best fit by a mono-exponential function (Equation 2).
D, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q 1 channels ( / 1 ratio, 0.3).
Recovery from ultra-slow inactivation was similar to DIV-A1529D I1303Q/F1304Q/M1305Q -only channels (compare with B).
Functional association of the 1 subunit with inactivation-defective DIV-A1529D subunits is confirmed by the acceleration of the current decay (compare with B).
9D I shows the growth of inward currents through DIV-A1529D 1303Q/F1304Q/M1305Q 1 channels with subsequent pulses at 20-s intervals.
Hence, coexpression of the 1 subunit with DIV-A1529D 1303Q/F1304Q/M1305Q channels did not affect ultra-slow inactivation.
DISCUSSION In this study, we investigated the relationship between fast inactivation and ultra-slow inactivation in DIV-A1529D channels.
First evidence in support for the existence of coupling between fast and ultra-slow inactivation came from experiments where we coexpressed the rat brain 1 subunit with DIVA1529D channels in Xenopus oocytes.
We found that coexpres- sion of the 1 subunit significantly delayed entry of DIVA1529D channels to the ultra-slow inactivated state during prolonged depolarizations (1).
In this study, we showed that coexpression of the 1 subunit exhibited similar modulatory effects on the current decay of DIV-A1529D and wild type 1 channels (Fig.
The 1 effects on DIV-A1529D channels (Table I) compare well with those of previous reports on 1 channels (22, 23).
This indicates that the functional association of 1 with the subunit was not disturbed by introducing the mutation DIV-A1529D.
Thus, coexpression of the 1 subunit does, as in 1 channels, stabilize the fast inactivated state in DIV-A1529D channels.
3, coexpression of the 1 subunit delayed entry to the ultra-slow inactivated state during prolonged depolarizations in DIV-A1529D channels.
The inhibition of ultra-slow inactivation by the 1 subunit in DIV-A1529D channels was directly Sodium Channel Inactivation Gate and Channel Structure related to the stabilization of the fast inactivated state since a negative linear correlation was found between the fraction of channels exhibiting a fast current decay and the fraction of channels that recovered from ultra-slow inactivation (Fig.
Thus, we wondered whether those DIV-A1529D-injected oocytes that in the absence of 1 showed a high amount of fast current decay also exhibited less ultra-slow inactivation than oocytes showing almost exclusively slow current decay.
If stabilization of the fast inactivated state inhibits ultraslow inactivation in DIV-A1529D channels, destabilization of the fast inactivated state would be expected to enhance ultraslow inactivation.
To destabilize the fast inactivated state, we used DIV-A1529D channels with the additional mutations I1303Q/F1304Q/M1305Q, which selectively remove fast inactivation (3, 4, 32).
Indeed, a considerable fraction of fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels could be driven to the ultra-slow inactivated state during a 300-s depolarization to 20 mV (Fig.
At this voltage, hardly any DIV-A1529D channels with normal fast inactivation properties entered the ultra-slow inactivated state most probably because they were fast inactivated.
Nevertheless, the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that entered the ultra-slow inactivated state after a 300-s prepulse to 20 mV, 0.44, was significantly smaller than the corresponding fraction after a 300-s prepulse to 50 mV, 0.58.
This indicated that stronger depolarizations modestly inhibited entry to the ultra-slow inactivated state also in fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
(1) we proposed that such a non-monotonic voltage dependence of ultra-slow inactivation in DIV-A1529D channels may arise if ultra-slow inactivation is reached via transitions from partially activated closed states that are passed through on the way up to the open state during activation.
Entry to the ultra-slow inactivated state from intermediate closed states is further corroborated by the fact that a considerable fraction of DIV-A1529D channels could be driven into ultra-slow inactivation by trains of brief depolarizations (Fig.
The fact that the fast inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels still exhibited a "residual" U-shaped voltage dependence of ultra-slow inactivation supports the notion that ultra-slow inactivation in this mutant is reached via transitions from pre-open states.
Whereas coexpression of the 1 subunit significantly reduced ultra-slow inactivation in DIV-A1529D channels, ultra-slow inactivation was not affected by 1 in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels (Fig.
Accordingly, in our experiments we observed an accelerated current decay in DIV-A1529D I1303Q/ F1304Q/M1305Q 1 channels (compare current traces of Fig.
Hence, 1 needs functional fast inactivation to be able to modulate ultra-slow inactivation in DIV-A1529D channels.
First, the addition of the selectivity filter mutation DIVA1529D to the inactivation gate mutation I1303Q/F1304Q/ M1305Q changed the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/M1305Q to a biexponential time course in DIV-A1529D I1303Q/F1304Q/ M1305Q (Fig.
This suggests that the mutation DIVA1529D created an additional inactivated state, i.e.
Second, as shown in a previous study, slow inactivation 37114 Sodium Channel Inactivation Gate and Channel Structure produced by 1-s prepulses in mutant DIV-A1529D had a monotonic voltage dependence, whereas the voltage dependence of ultra-slow inactivation was U-shaped, again arguing against direct transitions between slow inactivation and ultra-slow inactivation (1).
F1304Q
protein
substitution
P15390
true positive
This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/ I1303Q/F1304Q/ F1304Q/M1305Q.
1) and will subsequently be referred to as "ultraslow fraction." In the case of the mutant 1-I1303Q/F1304Q/M1305Q, which did not exhibit ultra-slow inactivation, recovery from slow inactivation was fit with the monoexponential function.
Mutagenesis of DIV-A1529D I1303Q/F1304Q/M1305Q--I1303Q/ F1304Q/M1305Q was made by oligonucleotide-directed mutagenesis and confirmed by sequencing the polymerized region.
DIV-A1529D was introduced in this construct by cloning the SacII-KpnI fragment into the I1303Q/F1304Q/M1305Q construct.
Effects of Destabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels The Inactivation-defective DIV-A1529D Channel Mutant I1303Q/F1304Q/M1305Q Enhances Ultra-slow Inactivation-- The strategy of all the experiments presented above was based on the stabilization of the fast inactivated state by different methods.
9 shows the growth of inward currents through inactivationdefective DIV-A1529D I1303Q/F1304Q/M1305Q channels with subsequent pulses at 20-s intervals.
Bi-exponential curve fits to the data points (solid lines, see "Experimental Procedures") were used to estimate the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that had entered the ultra-slow inactivated state after a 300-s prepulse to 50 and 20 mV.
Comparing these fractions between inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels and DIV-A1529D channels, it can be noticed that a prepulse potential of 50 mV produced similar fractions of channels recovering from ultra-slow inactivation in both cases, whereas a prepulse potential of 20 mV produced considerable ultra-slow inactivation only in inactivation-defective channels.
9C is the time course of recovery from a 300-s inactivating prepulse to 20 mV in I1303Q/F1304Q/M1305Q channels that did not carry the additional DIV-A1529D mutation in the selectivity filter (n 7).
The fact that adding the "selectivity filter mutation" DIV-A1529D to the "inactivation gate mutation" I1303Q/ F1304Q/M1305Q changes the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/ I M1305Q to a bi-exponential time course in DIV-A1529D 1303Q/F1304Q/M1305Q suggests that the mutation DIVA1529D created an additional inactivated state, i.e.
Stabilization of the fast inactivated state by a strongly depolarized potential ( 20 mV) dramatically inhibits entry to the ultra-slow inactivated state in DIV-A1529D channels but not in inactivation-defective DIV-A1529D I1303Q/F1304Q/ M1305Q channels (Fig.
In the latter, the fast inactivated state cannot be efficiently stabilized due to the mutations I1303Q/F1304Q/M1305Q.
If the fast inactivated state cannot be stabilized in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels, coexpression of the 1 subunit, which stabilizes the fast inactivated state in DIV-A1529D channels should not affect the fraction of DIV-A1529D I1303Q/F1304Q/M1305Q channels 37112 Sodium Channel Inactivation Gate and Channel Structure FIG.
Ultra-slow inactivation in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
A, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q.
For comparison, recovery from 300-s prepulses to 20 mV in the single mutant 1-I1303Q/ F1304Q/M1305Q is shown (n 7).
Whereas the time course of recovery in DIV-A1529D I1303Q/F1304Q/M1305Q (DIV-ADQQQ) was best fit by a bi-exponential equation (Equation 1), the time course of recovery in 1- I1303Q/F1304Q/M1305Q was best fit by a mono-exponential function (Equation 2).
D, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D I1303Q/F1304Q/M1305Q 1 channels ( / 1 ratio, 0.3).
Recovery from ultra-slow inactivation was similar to DIV-A1529D I1303Q/F1304Q/M1305Q -only channels (compare with B).
9D I shows the growth of inward currents through DIV-A1529D 1303Q/F1304Q/M1305Q 1 channels with subsequent pulses at 20-s intervals.
Hence, coexpression of the 1 subunit with DIV-A1529D 1303Q/F1304Q/M1305Q channels did not affect ultra-slow inactivation.
To destabilize the fast inactivated state, we used DIV-A1529D channels with the additional mutations I1303Q/F1304Q/M1305Q, which selectively remove fast inactivation (3, 4, 32).
Indeed, a considerable fraction of fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels could be driven to the ultra-slow inactivated state during a 300-s depolarization to 20 mV (Fig.
Nevertheless, the fraction of DIV-A1529D I1303Q/ F1304Q/M1305Q channels that entered the ultra-slow inactivated state after a 300-s prepulse to 20 mV, 0.44, was significantly smaller than the corresponding fraction after a 300-s prepulse to 50 mV, 0.58.
This indicated that stronger depolarizations modestly inhibited entry to the ultra-slow inactivated state also in fast inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels.
The fact that the fast inactivation-defective DIV-A1529D I1303Q/ F1304Q/M1305Q channels still exhibited a "residual" U-shaped voltage dependence of ultra-slow inactivation supports the notion that ultra-slow inactivation in this mutant is reached via transitions from pre-open states.
Whereas coexpression of the 1 subunit significantly reduced ultra-slow inactivation in DIV-A1529D channels, ultra-slow inactivation was not affected by 1 in inactivation-defective DIV-A1529D I1303Q/F1304Q/M1305Q channels (Fig.
Accordingly, in our experiments we observed an accelerated current decay in DIV-A1529D I1303Q/ F1304Q/M1305Q 1 channels (compare current traces of Fig.
Selective removal of fast inactivation by the mutation I1303Q/F1304Q/M1305Q allows slow inactivation to occur more quickly and completely (17, 33).
First, the addition of the selectivity filter mutation DIVA1529D to the inactivation gate mutation I1303Q/F1304Q/ M1305Q changed the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/M1305Q to a biexponential time course in DIV-A1529D I1303Q/F1304Q/ M1305Q (Fig.
12653681
full text
F193L
protein
substitution
true positive
P51787
Clinical Science (2003) 104, 377382 (Printed in Great Britain) 377 Clinical and electrophysiological characterization of a novel mutation (F193L) in the KCNQ1 gene associated with long QT syndrome Masato YAMAGUCHI*, Masami SHIMIZU*, Hidekazu INO*, Hidenobu TERAI*, Kenshi HAYASHI*, Hiroshi MABUCHI*, Naoto HOSHI and Haruhiro HIGASHIDA *Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8641, Japan, and Biophysical Genetics, Division of Neuroscience, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8641, Japan A B S T R A C T KCNQ1 is a gene encoding an subunit of voltage-gated cardiac K+ channels, with properties similar to the slowly activating delayed rectifier K+ current, and one of the genes causing long QT syndrome (LQTS).
We identified a novel mutation [Phe193Leu (F193L)] in the KCNQ1 gene in one family with LQTS.
Next, we studied the electrophysiological characteristics of the F193L mutation in the KCNQ1 gene using the expression system in Xenopus oocytes and the two-microelectrode voltage-clamp technique.
Co-expression of F193L KCNQ1 with the K+ channel minK suppressed peak (by 23.3 %) and tail (by 38.2 %) currents compared with those obtained by the combination of wildtype (WT) KCNQ1 and minK.
Time constants of current activation in F193L KCNQ1 and F193L KCNQ1jminK were significantly slower than those of WT KCNQ1 and WT KCNQ1jminK.
This electrophysiological study indicates that F193L causes less severe KCNQ1 current suppression, and thereby this mutation may result in a mildly affected phenotype.
Abbreviations : ECG, electrocardiogram ; I , slow component of the delayed rectifier K+ current ; LQTS, long QT syndrome ; Ks F193L, Phe193Leu ; JLNS, Jervell and Lange-Nielsen syndrome ; QTc, QT interval corrected for heart rate ; RFLP, restriction fragment length polymorphism ; SSCP, single-strand conformational polymorphism ; WT, wild-type.
A novel missense mutation [Phe193Leu (F193L)] in the S2S3 linker protein encoded by the KCNQ1 gene was identified.
In order to test the assumption of the relationship between the genotype and phenotype of this category of LQTS with in vitro evidence, we characterized the effect of the F193L mutation in the KCNQ1 gene on outward currents in Xenopus oocytes.
The F193L KCNQ1 cDNA was constructed by an overlap-extension strategy [15].
Wild-type (WT) KCNQ1 cDNA and F193L KCNQ1 cDNA were linearized by digestion with NotI, and cRNAs were prepared with the mMESSAGE mMACHINE kit (Ambion, Austin, TX, U.S.A.) using SP6 RNA polymerase.
Each oocyte was injected with 50 nl of cRNA containing 8.0 ng of WT KCNQ1 cRNA, 8.0 ng of F193L KCNQ1 cRNA, a combined cRNA composed of 8.0 ng of WT KCNQ1 and 1.0 ng of KCNE1, or a combined cRNA composed of 8.0 ng of F193L KCNQ1 and 1.0 ng of KCNE1.
Using a mutagenic primer, gene amplification by PCR introduced an artificial MvaI site into the PCR product only for the T C allele (F193L).
Characterization of an F193L mutation in the KCNQ1 gene 379 Statistical analysis All values are expressed as meanspS.E.M.
Sequence analyses revealed a heterozygous mutation leading to a single base substitution (T C) at position 739 in the KCNQ1 gene, resulting in an amino acid change from phenylalanine to leucine (F193L) (Figure 1A).
None of the carriers took any medication that might have affected Figure 1 Genetic analysis (A) Direct sequencing revealed a heterozygous nucleotide exchange (T739C) in the KCNQ1 gene that caused an amino acid exchange from phenylalanine to leucine at codon 193 (F193L).
#, no F193L mutation ; $, contain the F193L mutation.
The arrow shows the position of F193L.
Yamaguchi and others Figure 2 Expression study (A) Functional expression of WT KCNQ1 and F193L KCNQ1 alone or co-expressed with minK subunits in Xenopus oocytes.
Current traces represent typical examples of # 2003 The Biochemical Society and the Medical Research Society Characterization of an F193L mutation in the KCNQ1 gene 381 the characteristics of the ECG, or had previously experienced an episode of palpitation or syncope.
F193L KCNQ1 expression assay In the topology of the KCNQ1 subunit, the mutation identified was located in the intracellular linker of the S2S3 domains (Figure 1C).
To test this hypothesis, we examined the functional effect of the F193L mutation on the KCNQ1 subunit by using the Xenopus oocyte expression system.
Figure 2(A) shows the representative current traces recorded from those cells transiently transfected with 8 ng of WT KCNQ1 or F193L KCNQ1 without minK.
F193L KCNQ1 was able to form functional channels that resulted in a macroscopic outward current greater than non-injected oocytes, and smaller than WT KCNQ1.
Although co-expression of F193L KCNQ1 with minK also elicited similar outward currents, the peak and tail currents of mutants were smaller than those of WT KCNQ1 (Figure 2B).
Time constants of F193L KCNQ1 and F193L KCNQ1jminK were significantly slower than those of WT KCNQ1 and WT KCNQ1jminK (Figure 2C).
DISCUSSION The present study describes a novel missense mutation in the intracellular linker of the S2S3 domain of KCNQ1 (F193L) (Figure 1C), which did not show the dominantnegative effect on IKs functions.
By analysing functionality with WT or mutant clones in the absence or presence of minK in oocytes, we found that the F193L mutation in the KCNQ1 gene evoked an outward current with smaller amplitude than WT KCNQ1 gene.
(B) Currentvoltage relationships of the peak (upper) and tail (lower) currents measured at the end of, or after, the 2-s pulses for WT KCNQ1 (WT) and F193L KCNQ1 (F193L) in the absence or presence of minK.
(C) Time constants ( ) for slow and fast components of activation for WT KCNQ1 (WT) and F193L KCNQ1 (F193L) in the absence or presence of minK.
Yamaguchi and others In conclusion, we have identified at the molecular level and functionally characterized a novel mutation (F193L) in the intracellular S2S3 linker of KCNQ1 that is associated with a mild phenotype in a family.
R555C
protein
substitution
true positive
P51787
[22] found a missense mutation [Arg555Cys (R555C)] in the C-terminal domain of KCNQ1 in 44 patients from three congenital LQTS families.
11120735
full text
E1332P
protein
substitution
true negative
Note that the single channel conductances for barium and calcium are increased in the E1332P mutant.
E1332R
protein
substitution
true negative
Note that substitution of calcium for barium reduces peak current amplitude and slope conductance of wild type channels to a greater degree compared with the 1B (E1321K,D1323R,E1332R) mutant.
2, A and B displays macroscopic current-voltage relations of the wild type and the 1B (E1321K,D1323R,E1332R) triple mutant coexpressed in tsa-201 cells with the ancillary 1b and 2subunits.
To determine the relative contributions of the negatively charged residues in the putative external EF hand motif, we created double (E1321K,D1323R) and a single (E1332R) mutant channels and examined their relative permeabilities for barium and calcium at the single channel level.
A and B, single channel currentvoltage relations and current records obtained with wild type (WT) and E1321K,D1323R,E1332R mutant N-type calcium channels in either 100 mM barium or 100 mM calcium.
C and D, effect of double E1321K,D1323R and single E1332R substitutions on single channel conductance in barium and calcium.
E1321K
protein
substitution
true negative
Note that substitution of calcium for barium reduces peak current amplitude and slope conductance of wild type channels to a greater degree compared with the 1B (E1321K,D1323R,E1332R) mutant.
2, A and B displays macroscopic current-voltage relations of the wild type and the 1B (E1321K,D1323R,E1332R) triple mutant coexpressed in tsa-201 cells with the ancillary 1b and 2subunits.
To determine the relative contributions of the negatively charged residues in the putative external EF hand motif, we created double (E1321K,D1323R) and a single (E1332R) mutant channels and examined their relative permeabilities for barium and calcium at the single channel level.
A and B, single channel currentvoltage relations and current records obtained with wild type (WT) and E1321K,D1323R,E1332R mutant N-type calcium channels in either 100 mM barium or 100 mM calcium.
C and D, effect of double E1321K,D1323R and single E1332R substitutions on single channel conductance in barium and calcium.
G1326P
protein
substitution
true negative
4A shows single channel current-voltage relations obtained from the G1326P mutant in either 100 mM barium or 100 mM calcium.
The G1326P mutant displays similar single channel barium and calcium conductances.
B, bar graphs illustrating the single channel conductance obtained with the wild type (wt) channel and the G1326P mutant (coexpressed with 1b and 2- ) in either 100 mM barium or 100 mM calcium.
Inset, barium:calcium single channel conductance ratios for the wild type channel and G1326P mutant.
affect the basic biophysical properties of the channels, such as channel kinetics or the voltage dependences of activation and inactivation, and in particular the G1326P mutation did not significantly affect barium conductance of the channel.
Furthermore, the IC50 values for cadmium block of the triple E 0 utant (1.24 0.1 M, n 5) and the G1326P mutant (1.69 m .18 M, n 5) did not differ significantly (p 0.05) from that 5), obtained with the wild type channel (1.54 0.17 M, n indicating that the amino acid substitutions in the putative EF hand motif did not affect the narrow region of the pore.
D1323R
protein
substitution
true negative
Note that substitution of calcium for barium reduces peak current amplitude and slope conductance of wild type channels to a greater degree compared with the 1B (E1321K,D1323R,E1332R) mutant.
2, A and B displays macroscopic current-voltage relations of the wild type and the 1B (E1321K,D1323R,E1332R) triple mutant coexpressed in tsa-201 cells with the ancillary 1b and 2subunits.
A and B, single channel currentvoltage relations and current records obtained with wild type (WT) and E1321K,D1323R,E1332R mutant N-type calcium channels in either 100 mM barium or 100 mM calcium.
10625628
full text
D2S
protein
substitution
true negative
Potentiation of Kir3.1 3.2A Currents by Gi/o-coupled Receptors--We next investigated the issue of receptor specificity of channel activation by transiently transfecting a number of Gi/oand Gs-coupled receptors (A1 and A2A adenosine receptors, 2A and 1 adrenergic receptors, and D2S and D1 dopaminergic receptors, respectively) into the HKIR3.1/3.2 line.
The concentrations of agonists used in the present study (A1, A2A: 1 M NECA, 2A: 3 M noradrenaline, 1: 10 M isoprenaline, D2S: 10 M quinpirole, D1: 1 M SKF38393) were manyfold greater than KD values from radioligand binding studies published in the literature, such that receptors would exhibit full occupancy by agonist.
Stimulation of all three Gi/o-coupled receptors, A1, 2A, and D2S, potentiated currents and these increases were abolished by pre-treatment of the cells with PTx (Fig.
Maximal concentrations of agonists (1 M NECA (A1 and A2A), 3 M noradrenaline ( 2A), 10 M isoprenaline ( 1), 10 M quinpirole (D2S), and 1 M SKF38393 (D1)) were applied for 20 40 s.
Xu (D1 and D2S receptors) and the members of the HFSP collaboration for fruitful discussions (L.
C351I
protein
substitution
true negative
Point mutants of G i1 (C351G and C351I) were made as in Ref.
G i1C351G and G i1C351I, were co-expressed, agonist stimulation of receptor led to a large enhancement of currents (Fig.
A, this portion of the figure shows the effects of stimulating A1 receptors (1 M NECA) in PTx-treated cells (upper panel) and with the transfection of G i1C351G (middle panel) or G i1C351I (lower panel).
B, this bar chart summarizes the data obtained with the A1 receptor and G i1C351G, G i1C351I, and G s.
Mutation of Cys to either Gly (G i1C351G) or Ile (G i1C351I) was able to support coupling between both receptors and Kir3.1 3.2A.
C351G
protein
substitution
true negative
Point mutants of G i1 (C351G and C351I) were made as in Ref.
Transfection of of GTP S in the pipette solution enhanced basal current density from 103 exogenous G also increased current density from 112 17 pA/pF (n 41) to 701 226 pA/pF (n 14, p 0.01), in contrast to the reduction in current density observed with the transfection of subunits from Gi1 (41 6 pA/pF, n 10, p 0.05), Gi2 (20 3 pA/pF, n 10, p 0.01) or the PTx-insensitive mutant G i1C351G (34 8 pA/pF, n 8, p 0.05; see "Results").
G i1C351G and G i1C351I, were co-expressed, agonist stimulation of receptor led to a large enhancement of currents (Fig.
A, this portion of the figure shows the effects of stimulating A1 receptors (1 M NECA) in PTx-treated cells (upper panel) and with the transfection of G i1C351G (middle panel) or G i1C351I (lower panel).
B, this bar chart summarizes the data obtained with the A1 receptor and G i1C351G, G i1C351I, and G s.
Mutation of Cys to either Gly (G i1C351G) or Ile (G i1C351I) was able to support coupling between both receptors and Kir3.1 3.2A.
C1E
protein
substitution
true negative
The G Protein Subunit Has a Key Role in Determining the Specificity of Coupling to, but Not the Activation of, G Protein-gated Inwardly Rectifying K Channels* (Received for publication, August 2, 1999) F Joanne Louise Leaney, Graeme Milligan, and Andrew Tinker rom the Centre for Clinical Pharmacology, Department of Medicine, University College London, Rayne Institute, 5 University Street, London WC1E 6JJ and the Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, United Kingdom In neuronal and atrial tissue, G protein-gated inwardly rectifying K channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate.
10794678
full text
G285S
protein
substitution
P56696
true positive
A mutation in this gene in the DNFA2 pedigree exchanges the G for an S (G285S) in the GYG sequence in the pore of that channel, identical to the mutation in GIRK2wv.
The G285S mutation in KCNQ4 exerts a strong dominant negative effect on wild-type KCNQ4, and its loss leads to slow cellular degeneration (7), although the precise DIVALENT IMPERMEABILITY OF GIRK2WV K CHANNEL C1045 pathogenesis is unknown.
Unlike GIRK2wv, which has an equivalent mutation in the signature sequence, the mutation G285S in KCNQ4 does not appear to form functional homomultimers as does GIRK2wv.
Coexpression studies with the mutant KCNQ4 G285S and other members of the KCNQ family carried out to date show that coexpression of the mutant subunit reduces current expression by 90%.
G156S
protein
substitution
true positive
P48542
Am J Physiol Cell Physiol 278: C1038 C1046, 2000.--A single amino acid mutation (G156S) in the putative pore-forming region of the G proteinsensitive, inwardly rectifying K channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations.
MATERIALS AND METHODS THE MURINE WEAVER DISEASE is caused by the mutation of a single amino acid (G156S) in the putative pore region of the inwardly rectifying K channel, GIRK2.
12401812
full text
D7190G
protein
substitution
true negative
Voltage-gated Na currents were evoked by depolarizing pulses in HEK 293 cells transiently co-transfected with D7190G plus GFP or cotransfected with D1790G and FHF1B-GFP (Fig.
Typo
Y1795H
protein
substitution
true positive
Q14524
Fragments encoding point mutations (E1784K, D1790G, Y1795H, and Y1795C) and the multiple residue substitution (E1784K/D1790G/Y1795H) of the C terminus of Nav1.5 channel were also cloned into the pDBleu vector: E1784K (pDBNav1.5CK), D1790G (pDB-Nav1.5CG), Y1795H (pDB-Nav1.5CH), Y1795C (pDB-Nav1.5CC), and KGH (pDB-Nav1.5CKGH).
The position of the cardiac arrythmia mutants E1784K (32), D1790G (3335), and Y1795H and Y1795C (36) are shown.
A, schematic diagrams of wild type (Nav1.5C), naturally occurring point mutants (E1784K, D1790G, Y1795H, and Y1795C) and combination mutant (KGH).
These include E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
Y2H assays revealed that the mutations E1784K, Y1795H, or Y1795C did not affect FHF1B binding (Fig.
As might be expected, the triple mutant KGH (E1784K, D1790G, and Y1795H combined) did not show any interaction (Fig.
E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
Y1795C
protein
substitution
true positive
Q14524
Fragments encoding point mutations (E1784K, D1790G, Y1795H, and Y1795C) and the multiple residue substitution (E1784K/D1790G/Y1795H) of the C terminus of Nav1.5 channel were also cloned into the pDBleu vector: E1784K (pDBNav1.5CK), D1790G (pDB-Nav1.5CG), Y1795H (pDB-Nav1.5CH), Y1795C (pDB-Nav1.5CC), and KGH (pDB-Nav1.5CKGH).
The position of the cardiac arrythmia mutants E1784K (32), D1790G (3335), and Y1795H and Y1795C (36) are shown.
A, schematic diagrams of wild type (Nav1.5C), naturally occurring point mutants (E1784K, D1790G, Y1795H, and Y1795C) and combination mutant (KGH).
These include E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
Y2H assays revealed that the mutations E1784K, Y1795H, or Y1795C did not affect FHF1B binding (Fig.
E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
D1790G
protein
substitution
true positive
Q14524
Expression of Recombinant Na Channels--HEK 293 cells were grown under culture conditions and co-transfected with 1 g of wild type human Nav1.5 or its D1790G mutant cDNAs, together with 0.9 g of FHF1B-GFP and pEGFP-N1 cDNAs, using a calcium-phosphate protocol.
Fragments encoding point mutations (E1784K, D1790G, Y1795H, and Y1795C) and the multiple residue substitution (E1784K/D1790G/Y1795H) of the C terminus of Nav1.5 channel were also cloned into the pDBleu vector: E1784K (pDBNav1.5CK), D1790G (pDB-Nav1.5CG), Y1795H (pDB-Nav1.5CH), Y1795C (pDB-Nav1.5CC), and KGH (pDB-Nav1.5CKGH).
The mutant D1790G was generated using site-directed mutagenesis.
The forward mutagenic primer (5 -AGCCCCTGAGTGAGGACGGCTTCGATATGTTCTATG-3 ) and the reverse mutagenic primer (reverse complement sequence of the forward primer) were used in the QuikChange mutagenesis system (Stratagene); the single nucleotide substitution G, underlined and in bold type, causes the D1790G LQT-3 mutation.
The position of the cardiac arrythmia mutants E1784K (32), D1790G (3335), and Y1795H and Y1795C (36) are shown.
The LQT-3 point mutation D1790G in the C terminus of Nav1.5 abolishes the interaction between FHF1B and Nav1.5.
A, schematic diagrams of wild type (Nav1.5C), naturally occurring point mutants (E1784K, D1790G, Y1795H, and Y1795C) and combination mutant (KGH).
C, D1790G mutant abolishes the binding of FHF1B to Nav1.5 in vivo (co-immunoprecipitation assays).
Extracts prepared from HEK293 cells transfected with pFHF1B-GFP with either wild type Nav1.5 (lanes 3 and 4) or D1790G mutant (lanes 1 and 2) expression plasmids were incubated with antiGFP polyclonal antibodies (lanes 2 and 4).
These include E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
However, the D1790G mutant totally abolished the interaction with FHF1B (Fig.
As might be expected, the triple mutant KGH (E1784K, D1790G, and Y1795H combined) did not show any interaction (Fig.
The effect of the D1790G mutation on the interaction of FHF1B and full-length Nav1.5 in vivo was tested by an immunoprecipitation assay (Fig.
In this assay, extracts of cells expressing the mutant D1790G (lanes 1 and 2) or wild type human Nav1.5 (lanes 3 and 4) together with full-length FHF1B-GFP were first incubated with anti-GFP antibodies (lane 2 and 4), and the immunoprecipitated complexes were counter-tested with sodium channel pan-specific antibodies.
Both wild type Nav1.5 and the D1790G mutant channel were expressed efficiently as comparable levels of the channels were detected in the cell lysates (lanes 1 and 3).
6C, lane 4) but not the D1790G mutant (lane 2) co-immunoprecipitates with FHF1B-GFP.
Thus, FHF1B specifically binds to full-length Nav1.5 in vivo, and the LQT-3 mutation D1790G abolishes this interaction.
FHF1B Modulates Nav1.5 Channel Inactivation--The effects of FHF1B on voltage-dependent inactivation were examined by 1034 Interaction of FHF1B and Cardiac Sodium Channel functions were 40.27 3.15 mV and 8.68 0.59 mV/e-fold 8) and potential change, respectively, for D1790G (n 41.92 3.46 mV and 10.56 1.27 mV/e-fold potential change, respectively, for D1790G FHF1B (n 11).
Compared with the wild type Nav1.5 channels, the D1790G mutant channels show a depolarized shift of 10 mV in voltage-dependent activation (p 0.05) in agreement with published results (35).
Co-expression of FHF1B and D1790G did not modulate the voltage-dependent activation of the mutant channels.
The D1790G mutation causes a marked negative shift, 22 mV, in the voltage dependence of inactivation (Fig.
The average V1/2 and k values for the fitted functions were 98.74 2.82 mV and 10.82 1.06 mV/e-fold potential change, respectively, for D1790G mutant (n 16).
Co-expression of FHF1B with D1790G channels failed to affect voltagedependent inactivation of the channel.
The average V1/2 and k values for the fitted functions were 98.43 2.91 mV and 10.33 1.7 mV/e-fold potential change, respectively, for D1790G FHF1B (n 16).
The lack of modulation of D1790G channels by co-expression with FHF1B is consistent with the biochemical data showing that this mutation abolishes the binding of the proteins.
FHF1B does not modulate the D1790G mutant channel.
A, current traces recorded from HEK 293 cells co-transfected with D1790G and GFP.
C, normalized current-voltage relationships for D1790G (n 8) and D1790G FHF1B (n 11) are shown.
FHF1B Does Not Modulate D1790G Mutant Channel--Because LQT-3 point mutation D1790G abolishes the interaction between Nav1.5 channel and FHF1B in yeast two-hybrid and biochemical assays (Fig.
Voltage-gated Na currents were evoked by depolarizing pulses in HEK 293 cells transiently co-transfected with D7190G plus GFP or cotransfected with D1790G and FHF1B-GFP (Fig.
As seen in wild type Nav1.5 currents, co-expression of FHF1B protein with the D1790G mutant channel did not significantly enhance the peak current density.
The peak Na current densities (elicited by voltage pulses from 130 to 30 mV) were 0.17 0.06 nA/pF (n 17) for D1790G and 0.17 0.09 nA/pF for D1790G FHF1B (n 18, p 0.05), respectively.
The Na currents produced by the D1790G mutant channel showed faster inactivation kinetics (Fig.
Co-expression of FHF1B did not alter the kinetics of inactivation of D1790G currents (Fig.
The I-V relationships of D1790G and D1790G FHF1B were not significantly different (Fig.
Voltage-dependent activation of D1790G and D1790G FHF1B currents are shown in Fig.
The LQT-3 channel mutant D1790G abolishes the binding of Nav1.5 to FHF1B.
E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
(33) postulated that the D1790G mutation prevents the binding of 1 to Nav1.5 and causes a significant shift in the opposite direction.
Our results also show that the D1790G mutation abolishes the interaction with FHF1B.
If the FHF1B-mediated hyperpolarizing shift of the channel inactivation is caused by the recruit- 1035 ment of a kinase to the channel complex, the D1790G hyperpolarizing shift of channel inactivation might be produced by an alternate mechanism.
Because FHF1B binding to the Nav1.5 mimics the effects of D1790G mutation, it is possible that the level of expression of FHF1B can affect heart function.
E1784K
protein
substitution
true positive
Q14524
Fragments encoding point mutations (E1784K, D1790G, Y1795H, and Y1795C) and the multiple residue substitution (E1784K/D1790G/Y1795H) of the C terminus of Nav1.5 channel were also cloned into the pDBleu vector: E1784K (pDBNav1.5CK), D1790G (pDB-Nav1.5CG), Y1795H (pDB-Nav1.5CH), Y1795C (pDB-Nav1.5CC), and KGH (pDB-Nav1.5CKGH).
The position of the cardiac arrythmia mutants E1784K (32), D1790G (3335), and Y1795H and Y1795C (36) are shown.
A, schematic diagrams of wild type (Nav1.5C), naturally occurring point mutants (E1784K, D1790G, Y1795H, and Y1795C) and combination mutant (KGH).
These include E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
Y2H assays revealed that the mutations E1784K, Y1795H, or Y1795C did not affect FHF1B binding (Fig.
As might be expected, the triple mutant KGH (E1784K, D1790G, and Y1795H combined) did not show any interaction (Fig.
E1784K (32), D1790G (3335), and Y1795H and Y1795C (36).
11274414
full text
M11101K
protein
substitution
true negative
2 A, the RCA products from the 508, G542X, and M11101K probes were detected by Cy3-, FITC-, and Cy5-labeled decorator ODNs, respectively.
Typo
M1101K
protein
substitution
true positive
P13569
Physical mapping of three loci in the CFTR gene region by RCA Locus 508 ODN sequences Probe-primer PRP3 (89): 3 -CCCTCTTGACCTCGGAAGTCTCCCATTTTAATTCGTGTCACCTTCTTAAAtttt(CH2)18tttttACGTCATCATGAACATTACACGTTCCAC-3 C ircle3 (78): GTGGAACGTGTAATGTTCATGATGACGTGCATCCTTGACAGCCGATGAGGCTGGCATCCTTGACAGCCGATGAGGCTG Decorator probe: Det3-Cy3 (24): 5 -Cy3-GCATCCTTGACAGCCGATGAGGCT-3 Probe-primer PRP1 (89): 3 -GAACCTCTTCCACCTTAGTGTGACTCACCTCCAGTTGCTCGTTCTTAAAGtttt(CH2)18tttttATGATCACAGCTGAGGATAGGACATGCGA-3 C ircle1 (78): CGCATGTCCTATCCTCAGCTGTGATCATCAGAACTCACCTGTTAGACGCCACCAGCTCCAACTGTGAAGATCGCTTAT Decorator probe: Det1c-FITC (18): 5 -FITC-TCAGAACTCACCTGTTAG-3 ; Det1d-FITC (18): 5 -FITC-ACTGTGAAGATCGCTTAT-3 Probe-primer PRP4 (89): 3 -GACGGTTGACCAAGAACATGGACAGTTGTGACGCGACCAAGGTTTACTCTtttt(CH2)18tttttCTTGTACATGTCTCAGTAGCTCGTCAGT-3 C ircle4 (78): ACTGACGAGCTACTGAGACATGTACAAGGAGCAGTCCTGTCAGCTAGGTCACGGAGCAGTCCTGTCAGCTAGGTCACG Decorator probe: Det4-Cy5 (24): 5 -Cy5-GAGCAGTCCTGTCAGCTAGGTCACG-3 G542X M1101K Bold type: probe sequence; lowercase tttt(CH2)18ttttt: linker; standard type: RCA primer, circle, and decorator ODN sequences.
A mixture of 500 nM decorator probes (Det3-Cy3 for F508, Det1c-FITC, Det1d-FITC for G542X, Det4-Cy5 for M1101K) in 2 SSC, 1% BSA, and 0.1% Tween 20 was applied to the slides.
A mixture of 500 nM decorator probes (Det3-Cy3 for F508, Det1c-FITC, Det1d-FITC for G542X, Det4-Cy5 for M1101K) in 2 SSC, 1% BSA, and 0.1% Tween 20 was applied to the slides.
Instead, the P1 anchor ODN and the pair of P2 AD-ODNs for the G542X locus were c ohybridized w ith the 50-mer non-AD-ODN probes for the 508 and M1101K loci, which acted as reference markers.
(A) RCA detection of probes targeted to the G542X locus (FITC), the 508 locus (Cy-3), and the M1101K locus (Cy5) in normal human lymphocytes.
(C) Cohybridization of two PAC clones (extended green and red signals) with 508 (yellow), G542X (green), and M1101K (white) oligomer probes.
The overall RCA detection efficiency at the 508, G542X, and M1101K loci averaged 37%, 45%, and 35%, respectively.
The overall efficiency in detecting RCA signals at the 508, G542X, and M1101K loci were 48%, 44%, and 36% respectively.
Here, P1 and AD P2 ODNs were c ohybridized w ith the nonallele discriminating 508 and M1101K 50mer probes prev iously used for small t arget detection (Fig.
The w t 508 and M1101K loci were detected by using Cy3- and Cy5-labeled probes, respectively.
G542X
protein
substitution
true positive
P13569
Cell lines w ith mut ations at the G542X locus of the c ystic fibrosis transmembrane c onduct ance regulator (CFTR) gene (GM11497, heteroz ygous mut ation; GM11496; homoz ygous mut ations), a cell line (CTL2337) w ith a single C insertion bet ween nucleotides 5382 and 5383 of the BRCA1 gene, and st andard HeL a cell lines, were obt ained f rom the American Tissue Type Collection (Corriel Cell Repositories, Camden, NJ).
Physical mapping of three loci in the CFTR gene region by RCA Locus 508 ODN sequences Probe-primer PRP3 (89): 3 -CCCTCTTGACCTCGGAAGTCTCCCATTTTAATTCGTGTCACCTTCTTAAAtttt(CH2)18tttttACGTCATCATGAACATTACACGTTCCAC-3 C ircle3 (78): GTGGAACGTGTAATGTTCATGATGACGTGCATCCTTGACAGCCGATGAGGCTGGCATCCTTGACAGCCGATGAGGCTG Decorator probe: Det3-Cy3 (24): 5 -Cy3-GCATCCTTGACAGCCGATGAGGCT-3 Probe-primer PRP1 (89): 3 -GAACCTCTTCCACCTTAGTGTGACTCACCTCCAGTTGCTCGTTCTTAAAGtttt(CH2)18tttttATGATCACAGCTGAGGATAGGACATGCGA-3 C ircle1 (78): CGCATGTCCTATCCTCAGCTGTGATCATCAGAACTCACCTGTTAGACGCCACCAGCTCCAACTGTGAAGATCGCTTAT Decorator probe: Det1c-FITC (18): 5 -FITC-TCAGAACTCACCTGTTAG-3 ; Det1d-FITC (18): 5 -FITC-ACTGTGAAGATCGCTTAT-3 Probe-primer PRP4 (89): 3 -GACGGTTGACCAAGAACATGGACAGTTGTGACGCGACCAAGGTTTACTCTtttt(CH2)18tttttCTTGTACATGTCTCAGTAGCTCGTCAGT-3 C ircle4 (78): ACTGACGAGCTACTGAGACATGTACAAGGAGCAGTCCTGTCAGCTAGGTCACGGAGCAGTCCTGTCAGCTAGGTCACG Decorator probe: Det4-Cy5 (24): 5 -Cy5-GAGCAGTCCTGTCAGCTAGGTCACG-3 G542X M1101K Bold type: probe sequence; lowercase tttt(CH2)18ttttt: linker; standard type: RCA primer, circle, and decorator ODN sequences.
A mixture of 500 nM decorator probes (Det3-Cy3 for F508, Det1c-FITC, Det1d-FITC for G542X, Det4-Cy5 for M1101K) in 2 SSC, 1% BSA, and 0.1% Tween 20 was applied to the slides.
Human genomic DNA fibers were stretched f rom f reshly cultured nor mal peripheral blood ly mphoc y tes and GM11496 cells (homoz ygous G542X mut ation).
A mixture of 500 nM decorator probes (Det3-Cy3 for F508, Det1c-FITC, Det1d-FITC for G542X, Det4-Cy5 for M1101K) in 2 SSC, 1% BSA, and 0.1% Tween 20 was applied to the slides.
Ex periments designed for allele discrimination at the G542X locus did not use the PAC prehybridization step outlined above.
Instead, the P1 anchor ODN and the pair of P2 AD-ODNs for the G542X locus were c ohybridized w ith the 50-mer non-AD-ODN probes for the 508 and M1101K loci, which acted as reference markers.
The hybridization, ligation, RCA reactions, washes, and signal detection c onditions were carried out as described for the detection of G542X mut ations in interphase cells.
(A) RCA detection of probes targeted to the G542X locus (FITC), the 508 locus (Cy-3), and the M1101K locus (Cy5) in normal human lymphocytes.
(C) Cohybridization of two PAC clones (extended green and red signals) with 508 (yellow), G542X (green), and M1101K (white) oligomer probes.
abilit y of RCA to detect small genomic DNA sequences w ithin interphase nuclei and stretched DNA fibers we chose three 50-bp t argets at the 508, G542X, and M101K mut ation loci w ithin the CFTR gene.
2 A, the RCA products from the 508, G542X, and M11101K probes were detected by Cy3-, FITC-, and Cy5-labeled decorator ODNs, respectively.
The overall RCA detection efficiency at the 508, G542X, and M1101K loci averaged 37%, 45%, and 35%, respectively.
The overall efficiency in detecting RCA signals at the 508, G542X, and M1101K loci were 48%, 44%, and 36% respectively.
The sequences of the P1 and the t wo AD-P2 ODNs designed to detect mut ations at the G542X locus of the CFTR gene are given in Table 2.
Detection of the G542X mut ation also was done on stretched D N A f i b e r s p r e p a r e d f r o m n o r m a l l y m p h o c y t es a n d t h e GM11496 cell line homoz ygous for the G542X mut ation (Fig.
RCA detection of wt and mu alleles at the G542X locus of the CFTR gene.
(B) Discrimination of wt and mu alleles of the G542X locus on stretched DNA fibers.
The w t G542X allele is labeled w ith Cy5 while the mu allele is labeled w ith Cy3.
The merged image (Com) shows a yellow-blue-white hybridization pattern when the G542X allele is w t and a yellow-g reen-white pattern when G542X is a mu allele.
It was apparent, however, that w t and mu alleles at the G542X c ould be readily detected as many fibers show ing the hybridization profiles illustrated in Fig.
M101K
protein
substitution
true negative
abilit y of RCA to detect small genomic DNA sequences w ithin interphase nuclei and stretched DNA fibers we chose three 50-bp t argets at the 508, G542X, and M101K mut ation loci w ithin the CFTR gene.
C3383A
protein
substitution
true negative
line FF2914 c ont ain ing a single mut ation at the C3383A locus of the patched (Gorlin syndrome) gene was the gif t of A llen Bale (Yale Un iversit y).
RCA detection of wt and mu alleles at the A13073C locus of the p53 gene, the 5382C ins locus of the BRCA gene, and the C3383A locus of the patched (Gorlin syndrome) gene.
To deter mine whether mut ations c ould be detected in other genes, P1 P2 probe sets were prepared for the A13073C locus in p53, the 5382Cins locus in BRCA-1, and the C3383A locus in the patched gene (Table 2).
11055989
full text
12770942
full text
Y1206S
protein
substitution
true negative
doi:10.1038/sj.bjp.0705238 KATP channel openers (KCOs); mutant sulphonylurea receptor SUR2B(Y1206S); levcromakalim; rilmakalim; P1075; pinacidil; aprikalim; diazoxide; minoxidil sulphate; nicorandil GBC, glibenclamide; HEK cells, human embryonic kidney 293 cells; KATP channel, ATP-sensitive K+ channel; KCOs, KATP channel openers; Kir, inwardly rectifying K+ channel; NBD, nucleotide binding domain; P1075, N-cyano-N0 -(1,1-dimethylpropyl)-N00 -3-pyridylguanidine; SUR, sulphonylurea receptor Keywords: Abbreviations: Introduction ATP-sensitive K+ channels (KATP channels) are closed by intracellular ATP and opened by MgADP; hence, they link the metabolic state of the cell to membrane potential and excitability (Ashcroft & Ashcroft, 1990).
Methods Cell culture and transfection SUR2B(Y1206S) was constructed from murine SUR2B (GenBank D86038, Isomoto et al., 1996) using the QuikChange Site-Directed Mutagenesis System (Stratagene, Amsterdam, The Netherlands) as described (Hambrock et al., 2001).
Cells were transfected with the mammalian expression vector pcDNA3.1 (Invitrogen, Karlsruhe, Germany) containing the coding sequence of murine SUR2B or SUR2B(Y1206S) using lipofectAMINE and OPTIMEM (Invitrogen), and cell lines stably expressing these proteins were generated as described (Hambrock et al., 1998).
IC50 values were converted into inhibition constants, Ki, by correcting for the presence of the radioligand, L, according to the equation Ki IC50 1 L=KD 1 2 Results Opener binding to SUR2B(Y1206S) First, we wanted to examine to which degree the mutation Y1206S in SUR2B affected opener binding; in case of P1075, only a small decrease in KD by less than a factor of 2 was where KD is the equilibrium dissociation constant of the radioligand.
Russ et al 100 80 60 40 20 KATP channel openers in the absence of Mg2+ 371 [3H]P1075 + MgATP [3H]GBC + MgATP [3H]GBC - MgATP P1075 MXS [ H]Radioligand Binding (%BS) 0 100 80 60 40 20 0 RILMA NICO 3 100 80 60 40 20 0 10 9 8 7 6 5 4 3 10 9 8 7 6 5 4 3 LCRK DIAZ Opener (-log M) Figure 2 Binding of KCOs to SUR2B(Y1206S).
Therefore, the opener binding properties of SUR2B(Y1206S) were studied in the [3H]P1075 competition assay using KCOs selected from different structural groups (Figure 1); these experiments were conducted in the presence of MgATP (1 mM).
Table 1 Binding of KCOs to SUR2B(Y1206S) and SUR2B wild-type as seen in [3H]P1075 competition assays in the presence of MgATP (1 mM) Opener SUR2B(Y1206S) Ki (nM) SUR2Ba Ki (nM) (3.5, 5.5) (21, 26) (18, 26) (110, 120) (224, 468) (5900, 10,000) (38, 60) P1075 6.5 ()Pinacidil 46 Rilmakalim 20 Levcromakalim 148 Aprikalim 437 Diazoxide 17,000 72 Minoxidil sulphateb Nicorandil 7100 (6.2, 6.8) 4.4 (42, 50) 23 (15, 26) 21 (141, 155) 115 (417, 457) 324 (16,000, 18,000) 7800 (63, 83) 48 (6500, 7800) 9100 (6900, 12,000) [3H]GBC opener competition curves [3H]GBC binding to SUR is reduced by MgATP; in case of wild-type and mutant SUR2B this was because of a decrease in the number of binding sites by 75%, whereas the affinity remained unchanged (KD B22 and 4 nM for wild-type and mutant SUR2B, respectively; Loffler-Walz et al., 2002; Hambrock et al., 2002).
b Amplitudes were 7971 and 7272% BS for SUR2B(Y1206S) and wild-type, respectively.
Similar observations were made with the mutant channel, Kir6.2/SUR2B(Y1206S) (not shown).
Figure 5a shows a recording from a patch with good ATP Opener binding to SUR2B(Y1206S) as seen in [3H]GBC competition assays in the presence and absence of MgATP (1 mM) +MgATP A (%Bs) 4674 5474 3872 6272 4274 5072 3878 5775 4477 5676 100b 3073 5572 Ki (mM) 1.2 (1.1, 1.3) 6.6 (5.7, 7.6) 3.0 (1.7, 5.2) 38 (24, 60) 170 (160, 180) 330 (220, 500) At 300 mM:95% BS At 300 mM: 100% BS MgATP A (%Bs) 100 100 7971 100b 100b 100b nH 1.170.1 1.170.1 1.270.1 1.470.2 1.070.1 1.0c F F a Data for P1075 are from Hambrock et al.
Since the binding experiments were carried out with mutant SUR2B some electrophysiological experiments were performed also with the mutant channel, Kir6.2/SUR2B(Y1206S).
(a) Mutant channel: repetitive cumulative stimulation of the Kir6.2/SUR2B(Y1206S) channel by P1075 in the presence of 100 mM ATP and the absence of Mg2+.
Discussion Mutant vs wild-type SUR2B The experiments comparing mutant and wild-type SUR2B showed that the mutation Y1206S has no major effect on the affinity of opener binding nor on the channel opening action of P1075.
11711564
full text
Q159E
protein
substitution
true positive
O88704
Quadruple mutations (1CI137T, M141L, T157K, Q159E) made r 7.6-fold longer compared with 1C wild-type.
I137T
protein
substitution
O88704
true positive
However, 1CI137T (substitution of 1C Ile137 by Thr), 1CM141L and 1CT157K significantly made r 1.7-, 1.7- and 1.6-fold longer, respectively.
These effects were additive, and double mutations (1CI137T, M141L) made r 4-fold longer.
Quadruple mutations (1CI137T, M141L, T157K, Q159E) made r 7.6-fold longer compared with 1C wild-type.
There were three mutations (I137T, M141L and T157K) which significantly affected the activation kinetics, and the effects were 1.6- to 1.7-fold.
I137T introduced a polar but uncharged side chain.
T160N
protein
substitution
true positive
O88704
Image mutation
I140L
protein
substitution
true positive
O88704
Image mutation
S65A
protein
substitution
true negative
Functional expression and electrophysiological measurements All channel subunits and green fluorescent protein (GFP) S65A cDNA were subcloned into independent PCI vectors (Promega, Madison, WI, USA) and the mixture of vectors were transfected into COS-7 cells (RIKEN, Wako, Japan) using LipofectAMINE (Life Technologies, Inc.) as described before (Ishii et al.
T157K
protein
substitution
O88704
true positive
However, 1CI137T (substitution of 1C Ile137 by Thr), 1CM141L and 1CT157K significantly made r 1.7-, 1.7- and 1.6-fold longer, respectively.
Quadruple mutations (1CI137T, M141L, T157K, Q159E) made r 7.6-fold longer compared with 1C wild-type.
There were three mutations (I137T, M141L and T157K) which significantly affected the activation kinetics, and the effects were 1.6- to 1.7-fold.
A different number of charges was introduced only in T157K, with one positive charge gain.
M141L
protein
substitution
O88704
true positive
However, 1CI137T (substitution of 1C Ile137 by Thr), 1CM141L and 1CT157K significantly made r 1.7-, 1.7- and 1.6-fold longer, respectively.
These effects were additive, and double mutations (1CI137T, M141L) made r 4-fold longer.
Quadruple mutations (1CI137T, M141L, T157K, Q159E) made r 7.6-fold longer compared with 1C wild-type.
There were three mutations (I137T, M141L and T157K) which significantly affected the activation kinetics, and the effects were 1.6- to 1.7-fold.
M141L is a subtle conservative change.
V147I
protein
substitution
true positive
O88704
Image mutation
E158D
protein
substitution
true positive
O88704
Image mutation
9502794
full text
C629Y
protein
substitution
true positive
Q24270
T he AR66 al lele, car r y i ng the C629Y mutation, i s mai ntai ned as a h e t e r o z y g o u s s t o c k w i t h t h e C y O , w g 1en11 s e c o n d c h r o m o s o m e b a l a n c e r that car r ies an enhanc er trap transposon i nsert.
T he mutant chi mera DR1C629Y was made usi ng PCR mutagenesi s (C ormack, 1997) on a HpaI /SstI frag ment to convert the C629 codon TGT to a T y r codon (TAT).
RESULTS The AR66 mutation (C629Y) reduces dihydropyridinesensitive calcium channel currents in larval muscle Prev ious work has show n that the embr yonic lethal gene Dmca1D [formerly called l(2)35Fa] encodes an L -t y pe calcium channel 1 subunit in Drosophila (Eberl et al., 1998).
Analysis of C629 mutations in a heterologous expression system Because ver y little attention has been paid to the highly conser ved IS1 region, we have taken advantage of the clues prov ided by the C629Y mutation to focus attention on its role in calcium channel f unction using ex pression in Xenopus ooc y tes where current levels and interactions w ith aux iliar y subunits can be examined in a more controlled manner than i s possible w ith the in vivo muscle preparation.
When coex pressed w ith calcium channel 1b and 2 subunits, both the w ild-t y pe chimera (DR1) and the chimera carr y ing the C629Y mutation (DR1C629Y) gave detectable barium currents (Fig.
T hi s current reduction and slow ing of channel acti vation i s strongly remini scent of the in vivo phenot y pe of the C629Y mutation in lar val muscle.
CF, Ooc y tes were injected w ith 50 nl containing 1 chimera cRNA (C, F, w ild t y pe DR1; D, mutant DR1C629Y) plus 2 cRNA from rabbit skeletal muscle (Elli s et al., 1988) and 1b cRNA from rat brain (Pragnell et al., 1991).
D, Representati ve current traces from ooc y tes ex pressing the DR1 chimera carr y ing the mutation C629Y.
E, Peak inward current versus test potential (IV cur ves) for mutant (DR1C629Y) and w ild-t y pe (DR1) chimeras.
Functional characteri z ation of the C629Y mutation in domain IS1 using a Drosophila and rabbit cardiac 1 subunit chimera (DR1).
A, L ocation of the C629Y mutant change in the calcium channel 1 subunit.
T he position of the amino acid substitution (C629Y) in the AR66 allele i s indicated by the ar row.
T he mi ssense mutation (C629Y) i s found in a highly conser ved region of domain IS1 and i s caused by a substitution of A for G at nucleotide 1886 in the open reading frame (Z heng et al., 1995; Eberl et al., 1998).
T he top line shows the position of the C629Y mutation.
C ompared w ith the C629Y mutant, the w ild t y pe has faster acti vation kinetics at low current levels, and there i s no significant difference bet ween the t1/2 at low current (t1/2 6.1 0.4 msec; N 9) compared w ith that at high current (t1/2 5.8 0.2 msec) levels.
T he C629Y mutation a ffects the dihydropy ridine-sensiti ve D-current but not the amiloride-sensiti ve A-current in lar val muscle.
Open squares are w ild t y pe; closed triangles are mutant heteroz ygotes (C629Y/ ); and closed circles are mutant homoz ygotes (C629Y/C629Y ).
T he number of lar vae ( L) used and the number of muscle fibers ( F) recorded are L 9 and F 11 for w ild t y pe; L 8 and F 14 for mutant heteroz ygotes (C629Y/ ); and L 5 and F 8 for mutant homoz ygotes (C629Y/C629Y ).
E, Averaged D-t y pe barium current traces from w ild-t y pe (upper) and homoz ygous C629Y mutant (lower) muscle fibers from the ex periment in C.
C168G
protein
substitution
P15381
true positive
To gain f urther insight as to how changes at thi s site a ffect current levels and acti vation kinetics, we made additional mutants in which charge (C168D and C168K), si z e (C168G and C168W), and abilit y to form a di sulfide bond (C168S) were altered.
Replacing C ys w ith a smaller amino acid (C168G) had no dramatic effect on acti vation kinetics (Fig.
Ooc y tes were injected w ith truncated, w ild-t y pe 1C N60 (A, C) or one of the follow ing mutations in 1C N60: C168Y (B, D), C168S ( E), C168G ( F), and C168D ( G).
C168D
protein
substitution
P15381
true positive
To gain f urther insight as to how changes at thi s site a ffect current levels and acti vation kinetics, we made additional mutants in which charge (C168D and C168K), si z e (C168G and C168W), and abilit y to form a di sulfide bond (C168S) were altered.
Replacement w ith a negati vely charged residue (C168D) significantly reduced current (Fig.
Ooc y tes were injected w ith truncated, w ild-t y pe 1C N60 (A, C) or one of the follow ing mutations in 1C N60: C168Y (B, D), C168S ( E), C168G ( F), and C168D ( G).
C168K
protein
substitution
P15381
true positive
To gain f urther insight as to how changes at thi s site a ffect current levels and acti vation kinetics, we made additional mutants in which charge (C168D and C168K), si z e (C168G and C168W), and abilit y to form a di sulfide bond (C168S) were altered.
Nor were we able to record currents in C168K carr y ing a positi vely charged side chain at thi s site.
C168S
protein
substitution
P15381
true positive
To gain f urther insight as to how changes at thi s site a ffect current levels and acti vation kinetics, we made additional mutants in which charge (C168D and C168K), si z e (C168G and C168W), and abilit y to form a di sulfide bond (C168S) were altered.
T he C168S mutation shows current levels and acti vation kinetics ver y similar to that of the w ild t y pe (Fig.
However, the C168S change did produce a hy perpolari z ing shift in the IV cur ve (Fig.
Ooc y tes were injected w ith truncated, w ild-t y pe 1C N60 (A, C) or one of the follow ing mutations in 1C N60: C168Y (B, D), C168S ( E), C168G ( F), and C168D ( G).
M6X
protein
substitution
true negative
T he 1b construct (pCD 1) was made by inserting the 1.9 kb HindIII (blunted)/BamH I frag ment of rat brai n 1 (Prag nel l et al., 1991) i nto a PstI (blunted)/BglII cut vector pCDM6X L (Mar icq et al., 1991) that has a 5 -untranslated region (UT R) from the Xenopus -globin gene.
C168W
protein
substitution
P15381
true positive
T he C168W mutation was made by PCR mutagenesi s (C ormack, 1997).
To gain f urther insight as to how changes at thi s site a ffect current levels and acti vation kinetics, we made additional mutants in which charge (C168D and C168K), si z e (C168G and C168W), and abilit y to form a di sulfide bond (C168S) were altered.
We were not able to record currents in the mutant C168W carr y ing a bul k y side chain.
C168Y
protein
substitution
P15381
true positive
1 B), we chose the well-studied rabbit cardiac 1C to determine whether the equi valent mutation (C168Y) (Mikami et al., 1989) has effects similar to those seen in the DR1 chimera.
A s show n in Figure 3, A versus B, the currents are again dramatically reduced, and the time to reach hal f ma x imal amplitude i s slower in the mutant ( 1C N60 C168Y; t1/2 17.3 1.9 msec) than in the w ild t y pe ( 1C N60; t1/2 6.8 0.3 msec) (Fig.
T he C168Y change alters the si z e of the side chain.
To determine whether the reduction in current level and the slow ing of acti vation kinetics in the C168Y mutant was because of the di sruption 3 Fig ure 2.
Ooc y tes were injected w ith truncated, w ild-t y pe 1C N60 (A, C) or one of the follow ing mutations in 1C N60: C168Y (B, D), C168S ( E), C168G ( F), and C168D ( G).
D, Representati ve current traces from the mutant C168Y before (upper) and a fter (lower) treatment w ith 1 M ( )-Bay K 8644.
H, Peak current versus test potential (IV cur ves) for C168Y alone ( filled inver ted triangles), w ith 1 (open circles), and w ith 1 plus 1 M ( )-Bay K 8644 ( filled circles).
of 1 interaction, we compared the 1C N60C168Y mutant ex pressed w ith and w ithout the 1b subunit (Fig.
In addition, the ma x imum currents from the C168Y mutated channels are still enhanced 3.7-fold by the calcium channel agoni st ( )-Bay K 8644 (Fig.
T hus, the C168Y mutation does not block 1 interaction or stimulation by dihydropy ridine agoni sts.
11371446
full text
T449V
protein
substitution
true negative
Some channels, e.g., Y445A (above) and T449V (Melishchuk et al., 1998) do not progress beyond this stage.
D447N
protein
substitution
true negative
To characterize these abnormalities we examined gating and ionic currents generated by Shaker IR and by three nonconducting mutants, W434F, D447N, and Y445A, in 0 K .
W434F and D447N are similar to Shaker IR, showing Na and TMA permeability when dilated.
Channels were rendered nonconducting by one of three further mutations: W434F (Perozo et al., 1993), D447N (Olcese et al., 1994), or Y445A (Heginbotham et al., 1994).
Mutations were made by polymerase chain reaction between unique restriction sites Xba1/Bgl2 for W434F and Bgl2/Sma1 for D447N and Y445A.
Gating currents from all three mutants are similar in the presence of K and differ mainly in the deactivation rate of Ig OFF after large depolarizations, which is slowest in D447N and fastest in Y445A.
A Gating current changes upon removal of K fter removal of K internally and externally, two (D447N and W434F) of the three nonconducting mutants become defunct as judged from their gating currents.
The transition from normal to defunct proceeds only with pulsing (GomezLagunas, 1997) and, when the holding potential (HP) is 80 mV, requires roughly 100 gating cycles for W434F but only 5 for D447N.
1, B (W434F), C (Shaker IR), and D (D447N).
Shaker IR channels and the nonconducting W434F and D447N mutants have very similar gating currents in the defunct condition.
Q-V plots of three nonconducting mutants D447N, Y445A, and W434F are shown in the presence of potassium by the upper curves.
The lower curves are for Shaker IR, W434F, and D447N in the defunct condition.
Almost identical currents can be obtained for the D447N mutant (Fig.
(A) Shaker IR; (B) D447N; (C) Large-cation currents are simulated using the model given in the Discussion.
This is quite different from the behavior of Shaker, W434F, and D447N, which show a large TMA current (Fig.
o 3) An ionic current, carried by large cations like NMG r TMA , is found after K removal in channels that can become defunct (Shaker, W434F, and D447N) but not in Y445A, which cannot.
Others, e.g., Shaker (conducting) and W434F and D447N (both nonconducting), progress to the dilated condition in which large cations can permeate.
H4K
protein
substitution
true negative
MATERIALS AND METHODS Mutagenesis and expression Mutations were engineered in Shaker H4 K channel with the 6 46 mutation that removes N-type inactivation (Hoshi et al., 1990).
Y445A
protein
substitution
true negative
To characterize these abnormalities we examined gating and ionic currents generated by Shaker IR and by three nonconducting mutants, W434F, D447N, and Y445A, in 0 K .
Y445A does not become defunct and shows Na but not TMA permeability on K removal.
Channels were rendered nonconducting by one of three further mutations: W434F (Perozo et al., 1993), D447N (Olcese et al., 1994), or Y445A (Heginbotham et al., 1994).
Mutations were made by polymerase chain reaction between unique restriction sites Xba1/Bgl2 for W434F and Bgl2/Sma1 for D447N and Y445A.
Gating currents from all three mutants are similar in the presence of K and differ mainly in the deactivation rate of Ig OFF after large depolarizations, which is slowest in D447N and fastest in Y445A.
Y445A does not become defunct (cf.
Q-V plots of three nonconducting mutants D447N, Y445A, and W434F are shown in the presence of potassium by the upper curves.
Large-cation permeability was observed only in mutants that were subject to becoming defunct; i.e., all except Y445A (see below).
Y445A in 0 K i s Na b ut not TMA p ermeable Some mutant Shaker channels do not become defunct in 0 K (Melishchuk et al., 1998), although they demonstrate measurable sodium conductance.
6 shows currents of the nonconducting mutant Y445A elicited from HP 0 by the same voltage protocol as in Fig.
Y445A does not become defunct under any conditions we have found (see Fig.
Instead channels that have not been forced to close by a very Dilated Shaker Channels 2711 FIGURE 6 Y445A in 0 K solution does not become defunct and is permeable to Na but not to TMA .
(A) There is substantial ionic current through Y445A channels in 0 K when Na is present.
Y445A, another nonconducting mutant, does not become defunct.
o 3) An ionic current, carried by large cations like NMG r TMA , is found after K removal in channels that can become defunct (Shaker, W434F, and D447N) but not in Y445A, which cannot.
Some channels, e.g., Y445A (above) and T449V (Melishchuk et al., 1998) do not progress beyond this stage.
W434F
protein
substitution
true negative
To characterize these abnormalities we examined gating and ionic currents generated by Shaker IR and by three nonconducting mutants, W434F, D447N, and Y445A, in 0 K .
W434F and D447N are similar to Shaker IR, showing Na and TMA permeability when dilated.
Both Shaker IR and the nonconducting W434F mutant become measurably permer able to Na (Starkus et al., 1997, 1998) shortly after K emoval.
Channels were rendered nonconducting by one of three further mutations: W434F (Perozo et al., 1993), D447N (Olcese et al., 1994), or Y445A (Heginbotham et al., 1994).
Mutations were made by polymerase chain reaction between unique restriction sites Xba1/Bgl2 for W434F and Bgl2/Sma1 for D447N and Y445A.
A Gating current changes upon removal of K fter removal of K internally and externally, two (D447N and W434F) of the three nonconducting mutants become defunct as judged from their gating currents.
The transition from normal to defunct proceeds only with pulsing (GomezLagunas, 1997) and, when the holding potential (HP) is 80 mV, requires roughly 100 gating cycles for W434F but only 5 for D447N.
1, B (W434F), C (Shaker IR), and D (D447N).
1 A shows normal Ig (gating current) from the nonconducting W434F mutant in 30 K solution, using the pulse protocol 2706 Loboda et al.
Shaker IR channels and the nonconducting W434F and D447N mutants have very similar gating currents in the defunct condition.
Q-V plots of three nonconducting mutants D447N, Y445A, and W434F are shown in the presence of potassium by the upper curves.
The lower curves are for Shaker IR, W434F, and D447N in the defunct condition.
Trace 1 shows the normal gating current, typical of W434F, in 30 K solution.
4 B, closed circles) to those of W434F in 30 K solution (open circles), it is normal in shape and steepness, but left-shifted by 10 mV.
(A and B) Normal gating currents of W434F in 30 K solution (traces 1 and 4), the results of short (trace 2) and long (trace 5) exposure to 0 K solution (recorded in 0 K solution), and the currents after quick restoration of 30 K solution (traces 3 and 6).
Ig ON is quite small when the interruption is early (most of the channels are still dilated and nonconducting) but has a time course, when scaled up, that is indistinguishable from normal (i.e., W434F in 30 K solution).
(B) Q-V curves of normal (W434F in 30 K solution) and dilated channels.
This is quite different from the behavior of Shaker, W434F, and D447N, which show a large TMA current (Fig.
o 3) An ionic current, carried by large cations like NMG r TMA , is found after K removal in channels that can become defunct (Shaker, W434F, and D447N) but not in Y445A, which cannot.
Ba2 binds to the innermost site in the selectivity filter (Jiang and MacKinnon, 2000) and can prevent the W434F mutant from becoming defunct in the absence of K (Gomez-Lagunas, Dilated Shaker Channels 2713 1997; our observations).
Thus, the inner site is apparently intact and externally accessible in nonconducting mutants, implying that the lesion in W434F that destroys its conductance is a subtle alteration of the outer site.
This intimates that K occupancy of the inner site is crucial to maintain normal gating in W434F.
Others, e.g., Shaker (conducting) and W434F and D447N (both nonconducting), progress to the dilated condition in which large cations can permeate.
Macroscopic Na currents in the "nonconducting." Shaker potassium channel mutant W434F.
10990532
full text
F1613A
protein
substitution
true negative
Data analysis METHODS The class A Ca channel mutant 1AIF-AA was constructed by introducing two point mutations (I1612A and F1613A) into 1A cDNA (BI-2 accession number X57477, Mori et al.
Sequence difference
I1612A
protein
substitution
true negative
Data analysis METHODS The class A Ca channel mutant 1AIF-AA was constructed by introducing two point mutations (I1612A and F1613A) into 1A cDNA (BI-2 accession number X57477, Mori et al.
Sequence difference
11825900
full text
Y90F
protein
substitution
true positive
Q13303
To investigate the in vivo function of Kv 2, Kv 2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells.
Mice expressing Kv 2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype.
Kv 2-null mice and wild type littermates were 4258 days old, Kv 2-Y90F and wild type littermates were 50 53 days old, and Kv1.1-null mice (24) and wild type littermates were 30 41 days old.
onic stem cells to generate Kv 2-null or Y90F point mutant (which should specifically inhibit typical AKR catalytic activity) mice.
Construction of the Y90F Point Mutant Knock-in Vector Genomic DNA encoding exon 5 ( .
Creation of Targeted Embryonic Stem Cells and Kv 2-null and Y90F Mice AB-1 ES cells (obtained from Allan Bradley) were electroporated with 10 g of linearized targeting vector and treated with G418 (350 g/ml) and FIAU (200 nM) for selection of doubly resistant colonies.
For the ES cells transfected with the Kv 2-Y90F targeting vector, DNA was digested with BamHI (for the 3 probe) or AccI and AccI/SacII (for the neo probe).
The Kv 2-null and Y90F point mutant mice described in this report have been given the strain designations Kcnab2tm1Mes and Kcnab2tm2Mes, respectively.
Survival Analysis All Kv 2-null, Y90F, and heterozygous mice that were generated through the F2 and F3 generations (in mixed B6 129 backgrounds) were included in the survival analysis.
Electrophysiology Kv1.4 cRNA (30 pg) was coinjected with or without just-saturating amounts of Kv 2 or Kv 2-Y90F cRNA (60 pg) into Xenopus oocytes.
B, gene targeting for Kv 2 Y90F point mutant.
The exons are indicated by boxes (exon 5 containing the Y90F mutation is open), and the regions of DNA used for 5 and 3 arms of homology are indicated by thick black lines.
Introduction of the Y90F mutation into exon 5 also results in conversion of an AccI site to a SacII site.
Recombination at the 5 end was verified using the PstI-NotI fragment from neo as a probe and AccI for digestion, and inclusion of the Y90F mutation was verified by an AccI-SacII digestion.
In contrast, homozygous Kv 2-Y90F point mutants and heterozygous littermates showed no significant difference in mortality through 400 days.
TA B L E I Myoclonus scores Whole body myoclonus, similar to that seen in Kv1.1 null mice (24), is seen in Kv 2 nulls but not Y90F mutant mice.
Body weights (g S.E.) were comparable for each set of Kv mutants and their controls, although Kv1.1 mutants were slightly smaller than their wild type controls (for Kv1.1, wild type 15.0 0.5, mutant 12.1 0.7; for Kv 2, wild type 20.0 0 1.7, mutant 21.2 0.9; for Y90F, wild type 16.9 0.5, mutant 8.1 0.9).
Kv1.1-null Kv 2-null Kv 2-Y90F 0 (n 0 (n 0 (n 5) 9) 3) 86.25 24.11 24.40 (n 4) 8.12 (n 9) 0 (n 11) significant phenotypic consequences for the animal.
Electrophysiological Effects of Native and Mutant Y90F Kv 2 in Xenopus Oocytes--To investigate the potential enzymatic role of Kv 2 in vivo, we sought to create a mutation of the protein that would abolish enzymatic activity without altering the stability or potential nucleotide binding properties of the protein.
Kv 2-Y90F was subsequently coexpressed with human Kv1.4 channels in Xenopus oocytes as an assay for its stability and ability to interact with Kv1 channels (10).
Both native and mutant Y90F 2 subunit proteins accelerate the inactivation and increase the current amplitudes of Kv1.4 currents (Fig.
It also suggests that the Y90F 2 protein is stable.
Generation of Kv 2 Y90F Mice--To test the role of putative oxidoreductive catalysis by the Kv 2 subunit in vivo, mice containing the Y90F point mutation of the Kcnab2 gene were generated in two steps.
Mating F1 heterozygotes yielded the expected Mendelian ratios of wild type, heterozygous, and homozygous mutant mice ( / 2 22, Y90F/ 47, Y90F/Y90F 8; 2 0.84, df 2, p 0.66), as expected given the lack of embryonic lethality in the Kv 2-null mutation.
Expression of Y90F Kv 2 RNA transcripts was verified using reverse transcription-PCR in combination with AccI and SacII digests (data not shown).
Y90F Kv 2 Mice Do Not Show Phenotypic Alterations Observed in Kv 2-null Mutants--The Y90F mutant mice were not observed to undergo spontaneous seizures and were similar to wild type mice in both the swim test and life span analysis (Table I and Fig.
2; for life span analysis, Y90F/ n 21 and Gene Targeting of Kv 2 in Mice 13225 FIG.
Y90F/Y90F n 77).
DISCUSSION In this paper, we describe the generation of Kv 2-null and Kv 2-Y90F mutant mice to examine the in vivo function of Kv 2, a major auxiliary subunit of Kv1 channels in the mammalian nervous system.
Electrophysiological properties of coexpressed Kv1.4 and wild type or Kv 2-Y90F in Xenopus oocytes.
The straightforward question being asked is whether Y90F mice show any overt phenotype, and if so whether it is similar or identical to that observed for the Kv 2-null mice? The unequivocal answer to these questions is no.
In contrast, phenotypes potentially detectable in Y90F mice would not be limited in this way and could result from deficient catalysis of any cellular substrate(s).
Although more sophisticated testing will need to be performed before one can exclude an enzymatic role for the Kv 2 protein, given the discrepancy in phenotypes between the Kv 2-null and Y90F mutant mice, it appears that catalytic activity is not the primary physiological role of Kv 2 or that Kv 2 has atypical AKR enzymatic activity.
Whether mutations in Kv 2 other than Y90F at putative catalytic or cofactor binding sites (35, 43, 44) will reveal phenotypic effects in the mouse system remains to be established.
In addition, the lack of similar phenotypic effects in Kv 2-Y90F mice indicate that typical AKR oxidoreductive catalytic activity is unlikely to be the primary physiological role of Kv 2 gene products.
10608827
full text
Y628A
protein
substitution
true negative
MATERIALS AND METHODS Peptide Synthesis and Purification--rENaC constructs, including wt -rENaC and and subunits of Liddle's mutations R564 ( T), R574 ( T), Y618A ( Y), and Y628A ( Y) were kind gifts of Drs.
To test this hypothesis, subunits containing point mutations associated with Liddle's syndrome, Y618A ( Y) or Y628A ( Y), combined with the two complementary subunits of wt rENaC, were expressed in oocytes, and the effect of the peptides was examined.
Sequence different from P37089
Y618A
protein
substitution
true negative
MATERIALS AND METHODS Peptide Synthesis and Purification--rENaC constructs, including wt -rENaC and and subunits of Liddle's mutations R564 ( T), R574 ( T), Y618A ( Y), and Y628A ( Y) were kind gifts of Drs.
To test this hypothesis, subunits containing point mutations associated with Liddle's syndrome, Y618A ( Y) or Y628A ( Y), combined with the two complementary subunits of wt rENaC, were expressed in oocytes, and the effect of the peptides was examined.
Sequence different from P37089
R564X
protein
substitution
true negative
This finding was verified in oocytes expressing a rENaC construct containing a truncated version of the subunit (R564X).
Cell surface R564X -rENaC was increased over wt rENaC expression of expression, corresponding to a 5.6-fold increase in ENaC currents (25).
However, we previously reported that a 35% increase in the fluorescence intensity in oocytes injected with R564X -rENaC was accompanied by a 4.4-fold increase in amiloride-sensitive Na current (26).
Sequence different from P37089
11866477
full text
C121W
protein
substitution
true positive
Q00954
We have studied, in the HEK cells permanently transfected with the skeletal muscle sodium channel -subunit (SkM1), the effects of a transient transfection of the wild type (WT) or C121W mutant 1-subunit.
We have observed that mutant C121W lacks this modulatory property, but maintains its property to increase the current density.
Our observation suggests a possible involvement of this lack of modulation in the development of the GEFS , providing the first hypothesis based on the observation of the functional properties of the 1-subunit C121W mutant in mammalian cells, which certainly represents a more physiological preparation, instead of in Xenopus oocytes, where the modulatory properties of the 1-subunit are artificially amplified.
Surprisingly, however, no myopathic or cardiac symptoms have been reported in the family carrying the GEFS-linked C121W mutation [19], and the tissue specificity for this remains puzzling.
We have studied the effect of the epileptogenic mutation C121W of the 1-subunit on the sodium channel heterologously expressed in mammalian cells.
We observed that the mutation C121W does not affects the "facilitation" of sodium channel expression, but suppress the slight modulation of the 1-subunit on the sodium channel inactivation.
Transient expression of the WT or C121W 1-subunits was studied in HEK cells that permanently express the -subunit.
HEK- cells were transfected by electroporation with pRcBeta or pRcBetaC121W.
4, 2002 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 2 Comparison of the Electrophysiological Data Obtained from HEK Cells Permanently Transfected with -Subunit Alone 1) or Mutant ( 1-C121W) 1-Subunit ( ), and Cotransfected with Wild Type ( 1 1 (ms) rec( 120) (ms) rec( 140) (ms) V 0.5 (mV) V h (mV) 0.41 2.6 1.9 28.2 65.9 0.02 ( n 0.2 ( n 0.2 ( n 1.6 ( n 1.0 ( n 19) 7) 7) 22) 22) 0.40 1.9 1.1 30.3 68.9 0.02 ( n 0.2 ( n 0.2 ( n 1.7 ( n 1.2 ( n 24) 18) 18) 19) 19) 0.41 2.6 1.6 26.5 66.8 1-C121W 0.01 ( n 0.2 ( n 0.2 ( n 1.1 ( n 0.9 ( n 22) 14) 14) 23) 23) Note.
HEK- cells were co-transfected with pRcBeta (HEK- - 1/CD8) or pRcBetaC121W (HEK- - 1C121W/ CD8) together with pcI-CD8, to allow the identification of the effectively transfected cells.
Such interaction is beyond the scope of this paper, and we concentrated, therefore, on the comparison of the functional effects of wild type and C121W 1-subunits in the presence of the CD8-expression, using HEK- /CD8 cells as controls.
Differently, HEK- - 1C121W/CD8 cells were significantly smaller (C m 11.2 1.4 pF; n 23), and showed a parallel reduction of I PM to 1.8 0.2 nA.
Thus, the WT 1-subunit increases the expression of the sodium channels, as described for the oocyte preparation [7, 1214, 16] and for the mammalian expression system [10], and the C121W mutant of 1 maintains this property.
This is consistent with the observation that the C121W mutant RNA can be effectively translated into protein in a cell-free in vitro translation system [19] and with the observation that the deletion of a region near cysteine 121, that renders the 1-subunit nonfunctional in oocytes (unable to suppress the slow inactivation mode), does not eliminate its "competition" with the WT 1-subunit in a putative coassembly with the -subunit [21].
In general, there is not significant differences in half activation and inactivation potentials (Table 2) measured in the three groups, HEK- /CD8, HEK- - 1/CD8 and HEK- - 1C121W/CD8.
The inactivation kinetics does not change significantly by the coexpression of the wild type or the C121W 1-subunit (Figs.
Currents were evoked by a test potential of 0 mV in HEK- cotransfected with CD8 (A) are not different from HEK- cells co-transfected also with the wild type 1-subunit, HEK- - 1/CD8 (B) or with the C121W mutant of the 1-subunit, HEK- - 1C121W/CD8 (C).
Recovery from inactivation of HEK- - 1/CD8 (open squares), measured at 120 mV (D) and at 140 mV (E) is faster in HEK- - 1/CD8 than in HEK- - 1 (circles) HEK- - 1C121W/CD8 (filled squares).
3D3E), having for HEK- - 1C121W/CD8 values close to those of control.
The C121W mutation apparently maintain the property of the 1-subunit to increase the expression of the sodium channels, but appears to abol- ish its effects on the kinetics of recovery from inactivation, as also described for oocytes [19].
Finally, we did some experiments of transient transfection with the WT or C121W mutant 1-subunit cDNA cloned in frame cDNA coding the green florescent protein (GFP) we found more than 70% of cells showed clear spots of fluorescence when observed with an epi-illumination microscope, providing an additional prove of the high efficiency of expression of the 1-subunits.
The ability to revert the slow mode of inactivation is lost by the mutant C121W.
Since the single point mutation C121W has been correlated with the GEFS , the mechanism proposed for the pathology is, in analogy with the observation done in the oocytes, that the mutated 1subunit determines an abnormal high propensity of sodium channel slow inactivation mode, producing a hyper-excitable population of neurons, that would cause the epileptogenic focus [19].
We have confirmed that a small modification on the recovery from the inactivation by the wild type 1-subunit occurs also in mammalian cells, and this effect is abolished by the C121W mutation.
Since the abundant slow mode of inactivation observed in frog oocytes has rarely been observed in other, more physiological, preparation (cell lines or native cells), it is likely that the behavior of the 1-subunit and the C121W mutant in the oocyte expression system might be strictly preparation specific and therefore with poor physiological meaning.
Interestingly, both, WT and C121W mutant, when transfected transiently increased in the same extent the density of the sodium currents, suggesting that this mutation does not affect the putative role of the ancillary subunit in the expression and/or assembling of sodium channels.
We conclude that the 1-subunit has minor effects in the modulation of the functional properties of the sodium channel in cell lines, which perhaps become physiologically critical, as suggested by the correlation of the C121W mutation with a pathological condition.
9746514
full text
E1537A
protein
substitution
P15381
true positive
Moreover, inactivation of E1537A channels, in both Ca2 and Ba2 solutions, appeared to decrease with membrane depolarization, whereas inactivation of wild-type channels became faster with positive voltages.
E1537 mutant channels displayed slower inactivation kinetics in Ba2 and Ca2 solutions To investigate the possible role of the negatively charged E1537 residue in calcium-dependent inactivation, mutant channels E1537D, E1537Q, E1537S, E1537G, and E1537A were expressed in Xenopus oocytes.
2 shows typical whole-cell current recordings, for the wild-type and mutant E1537Q, E1537S, E1537A cardiac 1C calcium channels, i imax 1 1 Yo R zF Vm E0.5 exp T 1 ( 1) where i is the peak current obtained after a 5-s pulse to voltage Vm; imax is the peak current measured after a 5-s voltage pulse to 100 mV; and i/imax is their ratio when normalized to 1; Y0 is the fraction of noninactivating current; E0.5 is the midpotential of inactivation; z is the slope factor; and R, T, F have their usual meanings.
Wildtype 1C and mutant E1537Q, E1537S, E1537A were expressed in Xenopus oocytes with auxiliary 2b a nd 2a subunits.
In fact, all channels displayed a faster Ca2 dependent than Ba2 -dependent inactivation, as Ba2 currents invariably inactivated more slowly than the corre- sponding Ca2 traces, from the wild-type to the E1537A channel.
The slower Ba2 - and Ca2 -dependent inactivation in E1537A channels was also observed in the presence of the 3 subunit (Castellano et al., 1993) (results not shown), indicating that the reduced inactivation was independent of the nature of the subunit.
A careful examination of the whole-cell recordings nonetheless indicates that both Ba2 and Ca2 current traces inactivated faster for the wild-type than for the muE nt channels in the following order: wt ta E1537Q 1537S E1537A, thus suggesting that overall macroscopic inactivation was reduced for E1537 mutant channels.
Inactivation time con- TABLE 1 Whole-cell peak currents for wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, E1537G/ 2b / 2a, E1537Q/ 2b / 2a, and E1537S/ 2b / 2a channels in 10 mM Ba2 and 10 mM Ca2 solutions as the mean SEM of n independent experiments Wild-Type 10 Ba 10 Ca 1.3 0.61 0.2 (11) 0.08 (14) E1537A 0.87 0.43 0.06 (15) 0.05 (14) E1537G 0.38 0.25 0.06 (7) 0.03 (5) E1537Q 0.63 0.27 0.04 (24) 0.04 (11) E1537S 0.44 0.28 0.04 (8) 0.03 (7) Whole-cell currents were typically larger for the wild-type channel.
E1537 Mutations in Calcium Channel Inactivation 2a 1731 2a TABLE 2 Inactivation time constants for wild-type 1C/ 2b / 2a, E1537Q/ 2b / E1537A/ 2b / 2a channels recorded in 10 mM Ba2 or 10 mM Ca2 solutions w , E1537G/ 2b / i snact low , E1537S/ 2b / ( 2a , and Aslow Ca2 41 55 60 63 65 4% 6% 3% 5% 7% Channels 672 780 876 988 2501 B inact a (ms) f inact ast Ca2 ( ms) Afast Ca2 59 45 40 37 35 4% 6% 3% 5% 7% Ca2 ms) t 1C/ 2b / 2a E1537Q/ 2b / 2a E1537G/ 2b / 2a E1537S/ 2b / 2a E1537A/ 2b / 2a 43 (7) 26 (6)* 40 (5)** 40 (5)** 740 (5)** 49 64 69 55 56 2 (15) 10 (6)* 11 (5)* 9 (6)* 3 (12)* 496 640 701 712 2372 33 (15) 9 (6)*** 18 (5)*** 41 (6)*** 659 (12)*** Whole-cell current traces recorded at Vm 10 mV were fitted to single-exponential (Ba2 ) or double-exponential (Ca2 ) functions at time t 2 s (see Eq.
stants at Vm 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in ( the presence of 10 mM Ba2 (right panel) or 10 mM Ca2 left panel).
Ba2 inactivation time constants increased from 672 43 ms (n 7) for the wild-type 1C channel to 988 40 ms (n 5) for E1537S and to 2501 740 ms (n 5) for E1537A.
Indeed, ina ct for the Ba wild-type channel was different from the E1537Q channel time constant at the level p 0.1 (*), whereas they were significantly different at the level p 0.05 (**) for E1537G, E1537S, and E1537A channels.
faa sl ac inact The faster inactivation time constant fast was not significantly affected by mutations at E1537 as instct ranged from faa 49 2 ms (n 15) for the wild-type channel to 56 3 ms i (n 12) for E1537A.
On the other hand, the slower Ca2 nactivation time constant inowt increased from 496 33 ms sl ac (n 15) for the wild-type 1C channel to 712 41 ms (n 6 ) for E1537S and to 2378 660 ms (n 12) for E1537A.
The increase in inowt in the E1537A mutant was accompasl ac nied by a parallel increase in its relative importance as the relative amplitude of the slower Ca2 inactivation time constant increased from 41 4% for the wild-type channel to 65 7% for E1537A.
Inactivation time constants for Vm sl ac 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in the presence of 10 mM Ba2 (right) or 10 mM Ca2 (left).
Ba2 inactivation time constants increased from 672 43 ms (n 7) for the wild-type 1C channel to 988 40 ms (n 5) for E1537S and to 2501 740 ms (n 5) for E1537A.
The faster inactivation time constant instct was not significantly affected by mutations at E1537 as instct ranged from 49 faa faa 2 ms (n 15) for the wild-type 2 i channel to 56 3 ms (n 12) for E1537A.
On the other hand, the slower Ca nactivation time constant inowt increased from 496 33 ms (n 15) sl ac for the wild-type 1C channel to 712 41 ms (n 6) for E1537S and to 2378 660 ms (n 12) for E1537A.
Moreover, the relative amplitude of the slower Ca2 inactivation time constant increased from 41 4% for the wild-type channel to 65 7% for E1537A.
ina ct for faa Ba inact E1537Q was different at the level p 0.1 (*), and Ba for E1537G, E1537S, and E1537A was different at the level p 0.05 (**).
Furthermore, inowt in Ca2 sl ac inact 2 a nd Ba in Ba ppeared remarkably similar for all calcium channel combinations and were found to increase in parallel in E1537Q, E1537S, E1537G, and E1537A channels.
Whole-cell Ba2 traces were recorded at the peak voltage for the wild-type, E1537Q, and E1537A channels, normalized and superimposed to magnify the differences in inacc tivation kinetics.
4 A, E1537A Ba2 urrents did not appreciably decay over the 450-ms voltage pulse to 10 mV, in contrast to wild-type Ba2 currents.
Whole-cell traces shown in the inset suggests that the rate of current activation may also be reduced in E1537A channels with, on average, act 3.8 0.7 ms (n 12) as compared to act 2.1 0.8 ms (n 9) for the wild-type channel.
4 B) between the wild-type and the E1537A channel, because both activated around 25 mV and peaked at 0 mV.
This 8 mV shift was comparable to the shifts experimentally recorded for E1537Q (10 0.5 mV to 19 2 mV, n 11) and E1537A (0 0.5 mV to 6 2 mV, n 14) channels.
The macroscopic current expression was generally higher for the wild-type channel as whole-cell Ba2 currents averaged 1.3 0.2 A (n 11), whereas E1537Q and E1537A generated smaller Ba2 currents (see i also Table 1).
The first argument is circumstantial and pertains to the significant kinetic differences between E1537Q and E1537A, despite similar expression levels.
Indeed, for all Vm 10 mV, whole-cell Ba2 and Ca2 currents recorded for E1537Q were not significantly different in size than E1537A currents (Fig.
4 D, the superimposed yet not normalized whole-cell Ca2 current traces measured at Vm 10 mV for the wild-type, the E1537Q, and the E1537A channels yielded similar current amplitudes when recorded under the same experimental conditions.
Despite generating a identical peak current of 0.66 A, wild-type Ca2 traces nonetheless inactivated significantly faster than E1537A traces, as only 15% of the wild-type currents remained at the end of the 450-ms pulse compared to 70% of the E1537A currents.
E1537 Mutations in Calcium Channel Inactivation 1733 FIGURE 4 Whole-cell currents for wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, and E1537Q/ 2b / 2a channels were recorded in the presence of 10 mM Ba2 (upper panels) and 10 mM Ca2 (lower panels) by the pulse protocol previously described.
Ba2 inactivation was faster for wild-type E1537Q E1537A channels.
As shown in the insert, the E1537A activation also appeared to be noticeably slower.
The wild-type and E1537A current-voltage curves peaked at 0 mV, whereas the E1573Q channel peaked at 10 mV.
On average, I-V curves peaked at the following voltages: 6 2 mV (n 11) for the wild-type 1C/ 2b / 2a, 0 0.4 mV (n 15) for E1537A/ 2b / 2a, and 10 1 mV (n 14) for E1537Q/ 2b / 2a channels.
(C) Ba2 whole-cell current amplitude was higher on average for the wild-type channel with a peak current of 1.2 0.1 A (n 11) as compared to peak currents of 0.81 0.06 A (n 15) for E1537A/ 2b / 2a and 0.63 0.04 A (n 14) for a E1537Q/ 2b / 2a channels.
Current traces were not normalized because in that particular case, wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, and E1537Q/ 2b / 2a channels yielded whole-cell Ca2 currents in the same range.
As seen, wild-type 1C Ca2 currents were typically faster than E1537A Ca2 currents, independently of the whole-cell current amplitude.
Wild-type and E1537Q Ca2 currents peaked, respectively, at Vm 14 1 mV (n 14) and 19 3 mV (n 11), whereas E1537A Ca2 currents peaked at Vm 5 1 mV (n 14).
(F) Whole-cell Ca2 current amplitude was higher on average for the wild-type channel with a peak current of 0.61 0.08 A (n 14) as compared to peak currents of 0.43 0.05 A (n 14) for E1537A/ 2b / 2a and 0.27 0.04 A (n 11) for E1537Q/ 2b / 2a channels.
Voltage dependence of the E1537A inactivation time constants In voltage-dependent ion channels, voltage controls kinetic transitions, and more specifically, positive voltage encour- ages transitions from the closed to the open state, and ultimately to the inactivated state.
We thus investigated the influence of voltage on the inactivation time constants for the E1537A mutant channel.
Inactivation time constants for the wild-type 1C/ 2b / 2a 2nd E1537A/ 2b / 2a channels were estimated at time t a s and reported as a function of the applied membrane potential between 10 and 20 mV (Fig.
As seen in the left panel, ina ct for the wild-type channel decreased from Ba 918 43 ms to 627 46 ms (n 7) between 10 and 20 mV; hence membrane depolarization appeared to 1734 Biophysical Journal Volume 75 October 1998 FIGURE 5 Inactivation time constants for the wild-type 1C/ 2b / 2a and E1537A/ 2b / 2a channels were estimated between time t 0 and time t 2 s and reported as a function of the applied membrane potential between 10 and 20 mV.
In contrast, the E1537A inactivation time constants increased at least threefold over the same voltage range with ina ct Ba 1221 246 ms at 10 mV to 3609 1329 ms (n 5) at 20 mV.
Note that the inactivation time constants estimated at 10 mV are not significantly different for the wild-type and the E1537A channel.
Again, the fast Ca2 inactivation time constant is not significantly different between the wild-type and the E1537A channel, with instct ranging from 41 5 ms faa to 73 5 ms (n 7) for the wild-type channel and 49 3 ms to 69 4 ms (n 10) for E1537A.
In contrast, the slow Ca2 inactivation time constant 7inowt was consistently higher for E1537A than for wild-type 1C channels at all membrane potentials.
Thus the E1537A channel appeared to become slower in response to membrane depolarization in both Ba2 and Ca2 solutions.
In contrast, the E1537A inactivation time constants increased Ba at least threefold over the same voltage range, with ina ct increasing from 1221 246 ms at 10 mV to 3609 1329 ms (n 5) at 20 mV.
Note that the inactivation time constants estimated at 10 mV were not significantly different for the wild-type and the E1537A channel.
3, instct was not significantly different between the wild-type and the E1537A channel, with instct ranging faa from 41 5 ms to 73 5 ms (n 7) for the wild-type channel and from 49 3 ms to 69 4 ms (n 10) for sl ac E1537A.
In contrast, inowt in Ca2 was consistently higher for E1537A than for wild-type 1C channels at all memsl ac brane potentials.
For the wild-type channel, inowt decreased from 732 7 ms to 521 51 ms (n 7), whereas in E1537A channels, inowt actually increased from 990 sl ac 99 ms to 1823 115 ms (n 10) between 10 and 20 mV.
Thus, not only was the E1537A channel slower at all membrane potentials than the wild-type channel; it also appeared to become slower with membrane depolarization in both Ba2 and Ca2 solutions.
E1537 Mutations in Calcium Channel Inactivation 1735 tigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels.
6 shows the whole-cell current traces recorded for wild-type 1C/ 2b / 2a and E1537A/ 2b / 2a channels by ( the tripulse protocol shown, in the presence of 10 mM Ba2 upper panel) and 10 mM Ca2 (lower panel).
In contrast, only 11 4% (n 6) of the E1537A channels were com- pletely inactivated under the same conditions.
The E1537A inactivation in Ba2 was so shallow that it could not be approximated by Boltzmann functions.
After a 5-s voltage pulse of 10 mV, whole-cell wild-type currents were inactivated at 83 5% (n 11), as 1 compared to 75 2% (n 4) of the E1537Q currents, 80 % (n 4) of the E1537G currents, and 81 5% (n 4) of FIGURE 6 The voltage dependence of inactivation was investigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels at the end of a 5-s prepulse.
Only the whole-cell current traces obtained with the wild-type and the E1537A channels are shown.
In contrast, only 11 4% (n 6) of the E1537A channels (E) were completely inactivated under the same conditions.
The E1537A inactivation data point could not be approximated by Boltzmann functions.
With its inactivation level of 69 3% (n 7), E1537A inactivation data points were almost indistinguishable from the wild-type and other E1537 mutants.
The fit parameters were z 2.8 0.2 and E0.5 s 22 1 mV (wild-type); z 1.9 0.2 and E0.5 18 2 mV (E1537G); z 1.9 .2 and E0.5 16 1 mV (E1537S); z 1.6 0.4 and E0.5 22 3 mV (E1537Q); z 3.0 0.3 and E0.5 17.0 0.7 mV (E1537A).
With a inactivation level of 69 3% (n 7), E1537A yielded inactivation data points in Ca2 that were almost indistinguishable from the wild-type and other E1537 mutants.
Isochronic inactivation experiments performed at a shorter time, t 2 s, proved to be qualitatively similar, with the exception that inactivation was somewhat reduced in E1537A channels with 49 3% (n 3) (results not shown).
Five-second pulses to positive membrane potentials in Ba2 failed to inactivate more than 15% of the E1537A channels, in contrast to 70% of the wild-type channels.
Mutant channels E1537Q, E1537S, E1537G, and E1537A were found to display significantly slower inactivation kinetics in Ba2 and Ca2 solutions.
E1537 Mutations in Calcium Channel Inactivation 1737 E1537A D1546A (Zhou et al., 1997).
The triple mutant failed to abolish calcium-dependent inactivation, although the Ca2 current traces they recorded from the 1C triple mutant (D1535A E1537A D1546A) were actually twice as slow as the ones recorded for the wild-type channel.
When a similar analysis was applied to our E1537 mutant data, we found that the calcium sensitivity factor f would decrease somewhat from the wild-type to the E1537A channel from 0.68 0.09 (n 4) to the nonzero value of 0.41 0.03 (n 5).
However, we cannot exclude a small effect on calciumdependent inactivation, as the extent of "steady-state" calcium-dependent decreased slightly from 85% to 70% from the wild-type to the E1537A mutant.
E1573Q
protein
substitution
true negative
The wild-type and E1537A current-voltage curves peaked at 0 mV, whereas the E1573Q channel peaked at 10 mV.
Typo
E1537D
protein
substitution
P15381
true positive
E1537 mutant channels displayed slower inactivation kinetics in Ba2 and Ca2 solutions To investigate the possible role of the negatively charged E1537 residue in calcium-dependent inactivation, mutant channels E1537D, E1537Q, E1537S, E1537G, and E1537A were expressed in Xenopus oocytes.
Except for the E1537D mutant, for which we never got expression, all E1537 mutant yielded measurable Ba2 and Ca2 currents with bona fide Ca2 channel characteristics (see also later in Fig.
E1537G
protein
substitution
P15381
true positive
E1537 mutant channels displayed slower inactivation kinetics in Ba2 and Ca2 solutions To investigate the possible role of the negatively charged E1537 residue in calcium-dependent inactivation, mutant channels E1537D, E1537Q, E1537S, E1537G, and E1537A were expressed in Xenopus oocytes.
This observation also applied to E1537G wholecell currents that are not shown in this figure.
Inactivation time con- TABLE 1 Whole-cell peak currents for wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, E1537G/ 2b / 2a, E1537Q/ 2b / 2a, and E1537S/ 2b / 2a channels in 10 mM Ba2 and 10 mM Ca2 solutions as the mean SEM of n independent experiments Wild-Type 10 Ba 10 Ca 1.3 0.61 0.2 (11) 0.08 (14) E1537A 0.87 0.43 0.06 (15) 0.05 (14) E1537G 0.38 0.25 0.06 (7) 0.03 (5) E1537Q 0.63 0.27 0.04 (24) 0.04 (11) E1537S 0.44 0.28 0.04 (8) 0.03 (7) Whole-cell currents were typically larger for the wild-type channel.
E1537 Mutations in Calcium Channel Inactivation 2a 1731 2a TABLE 2 Inactivation time constants for wild-type 1C/ 2b / 2a, E1537Q/ 2b / E1537A/ 2b / 2a channels recorded in 10 mM Ba2 or 10 mM Ca2 solutions w , E1537G/ 2b / i snact low , E1537S/ 2b / ( 2a , and Aslow Ca2 41 55 60 63 65 4% 6% 3% 5% 7% Channels 672 780 876 988 2501 B inact a (ms) f inact ast Ca2 ( ms) Afast Ca2 59 45 40 37 35 4% 6% 3% 5% 7% Ca2 ms) t 1C/ 2b / 2a E1537Q/ 2b / 2a E1537G/ 2b / 2a E1537S/ 2b / 2a E1537A/ 2b / 2a 43 (7) 26 (6)* 40 (5)** 40 (5)** 740 (5)** 49 64 69 55 56 2 (15) 10 (6)* 11 (5)* 9 (6)* 3 (12)* 496 640 701 712 2372 33 (15) 9 (6)*** 18 (5)*** 41 (6)*** 659 (12)*** Whole-cell current traces recorded at Vm 10 mV were fitted to single-exponential (Ba2 ) or double-exponential (Ca2 ) functions at time t 2 s (see Eq.
stants at Vm 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in ( the presence of 10 mM Ba2 (right panel) or 10 mM Ca2 left panel).
Indeed, ina ct for the Ba wild-type channel was different from the E1537Q channel time constant at the level p 0.1 (*), whereas they were significantly different at the level p 0.05 (**) for E1537G, E1537S, and E1537A channels.
Inactivation time constants for Vm sl ac 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in the presence of 10 mM Ba2 (right) or 10 mM Ca2 (left).
ina ct for faa Ba inact E1537Q was different at the level p 0.1 (*), and Ba for E1537G, E1537S, and E1537A was different at the level p 0.05 (**).
Furthermore, inowt in Ca2 sl ac inact 2 a nd Ba in Ba ppeared remarkably similar for all calcium channel combinations and were found to increase in parallel in E1537Q, E1537S, E1537G, and E1537A channels.
E1537 Mutations in Calcium Channel Inactivation 1735 tigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels.
After a 5-s voltage pulse of 10 mV, whole-cell wild-type currents were inactivated at 83 5% (n 11), as 1 compared to 75 2% (n 4) of the E1537Q currents, 80 % (n 4) of the E1537G currents, and 81 5% (n 4) of FIGURE 6 The voltage dependence of inactivation was investigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels at the end of a 5-s prepulse.
1): z 2.4 0.2, E0.5 20 0.7 mV (wild-type); z 1.4 0.1, E0.5 18 2 mV (E1537Q), z 1.2 0.2, E0.5 19 1 mV (E1537S); z 1.3 0.1, E0.5 17 2 mV (E1537G).
At 10 mV, whole-cell wild-type currents (F) were inactivated at 83 5% (n 11), as compared to the inactivation level of 75 2% (n 4) for the E1537Q currents (j), 80 1% (n 4) for E1537G (,), 81 5% (n 4) for E1537S ().
The fit parameters were z 2.8 0.2 and E0.5 s 22 1 mV (wild-type); z 1.9 0.2 and E0.5 18 2 mV (E1537G); z 1.9 .2 and E0.5 16 1 mV (E1537S); z 1.6 0.4 and E0.5 22 3 mV (E1537Q); z 3.0 0.3 and E0.5 17.0 0.7 mV (E1537A).
Mutant channels E1537Q, E1537S, E1537G, and E1537A were found to display significantly slower inactivation kinetics in Ba2 and Ca2 solutions.
D1546A
protein
substitution
P15381
true positive
E1537 Mutations in Calcium Channel Inactivation 1737 E1537A D1546A (Zhou et al., 1997).
The triple mutant failed to abolish calcium-dependent inactivation, although the Ca2 current traces they recorded from the 1C triple mutant (D1535A E1537A D1546A) were actually twice as slow as the ones recorded for the wild-type channel.
E1537Q
protein
substitution
P15381
true positive
E1537 mutant channels displayed slower inactivation kinetics in Ba2 and Ca2 solutions To investigate the possible role of the negatively charged E1537 residue in calcium-dependent inactivation, mutant channels E1537D, E1537Q, E1537S, E1537G, and E1537A were expressed in Xenopus oocytes.
2 shows typical whole-cell current recordings, for the wild-type and mutant E1537Q, E1537S, E1537A cardiac 1C calcium channels, i imax 1 1 Yo R zF Vm E0.5 exp T 1 ( 1) where i is the peak current obtained after a 5-s pulse to voltage Vm; imax is the peak current measured after a 5-s voltage pulse to 100 mV; and i/imax is their ratio when normalized to 1; Y0 is the fraction of noninactivating current; E0.5 is the midpotential of inactivation; z is the slope factor; and R, T, F have their usual meanings.
Wildtype 1C and mutant E1537Q, E1537S, E1537A were expressed in Xenopus oocytes with auxiliary 2b a nd 2a subunits.
A careful examination of the whole-cell recordings nonetheless indicates that both Ba2 and Ca2 current traces inactivated faster for the wild-type than for the muE nt channels in the following order: wt ta E1537Q 1537S E1537A, thus suggesting that overall macroscopic inactivation was reduced for E1537 mutant channels.
Inactivation time con- TABLE 1 Whole-cell peak currents for wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, E1537G/ 2b / 2a, E1537Q/ 2b / 2a, and E1537S/ 2b / 2a channels in 10 mM Ba2 and 10 mM Ca2 solutions as the mean SEM of n independent experiments Wild-Type 10 Ba 10 Ca 1.3 0.61 0.2 (11) 0.08 (14) E1537A 0.87 0.43 0.06 (15) 0.05 (14) E1537G 0.38 0.25 0.06 (7) 0.03 (5) E1537Q 0.63 0.27 0.04 (24) 0.04 (11) E1537S 0.44 0.28 0.04 (8) 0.03 (7) Whole-cell currents were typically larger for the wild-type channel.
E1537 Mutations in Calcium Channel Inactivation 2a 1731 2a TABLE 2 Inactivation time constants for wild-type 1C/ 2b / 2a, E1537Q/ 2b / E1537A/ 2b / 2a channels recorded in 10 mM Ba2 or 10 mM Ca2 solutions w , E1537G/ 2b / i snact low , E1537S/ 2b / ( 2a , and Aslow Ca2 41 55 60 63 65 4% 6% 3% 5% 7% Channels 672 780 876 988 2501 B inact a (ms) f inact ast Ca2 ( ms) Afast Ca2 59 45 40 37 35 4% 6% 3% 5% 7% Ca2 ms) t 1C/ 2b / 2a E1537Q/ 2b / 2a E1537G/ 2b / 2a E1537S/ 2b / 2a E1537A/ 2b / 2a 43 (7) 26 (6)* 40 (5)** 40 (5)** 740 (5)** 49 64 69 55 56 2 (15) 10 (6)* 11 (5)* 9 (6)* 3 (12)* 496 640 701 712 2372 33 (15) 9 (6)*** 18 (5)*** 41 (6)*** 659 (12)*** Whole-cell current traces recorded at Vm 10 mV were fitted to single-exponential (Ba2 ) or double-exponential (Ca2 ) functions at time t 2 s (see Eq.
stants at Vm 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in ( the presence of 10 mM Ba2 (right panel) or 10 mM Ca2 left panel).
Indeed, ina ct for the Ba wild-type channel was different from the E1537Q channel time constant at the level p 0.1 (*), whereas they were significantly different at the level p 0.05 (**) for E1537G, E1537S, and E1537A channels.
Inactivation time constants for Vm sl ac 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in the presence of 10 mM Ba2 (right) or 10 mM Ca2 (left).
ina ct for faa Ba inact E1537Q was different at the level p 0.1 (*), and Ba for E1537G, E1537S, and E1537A was different at the level p 0.05 (**).
Furthermore, inowt in Ca2 sl ac inact 2 a nd Ba in Ba ppeared remarkably similar for all calcium channel combinations and were found to increase in parallel in E1537Q, E1537S, E1537G, and E1537A channels.
Whole-cell Ba2 traces were recorded at the peak voltage for the wild-type, E1537Q, and E1537A channels, normalized and superimposed to magnify the differences in inacc tivation kinetics.
The E1537Q peaked at 10 mV in the presence of Ba2 .
This 8 mV shift was comparable to the shifts experimentally recorded for E1537Q (10 0.5 mV to 19 2 mV, n 11) and E1537A (0 0.5 mV to 6 2 mV, n 14) channels.
The macroscopic current expression was generally higher for the wild-type channel as whole-cell Ba2 currents averaged 1.3 0.2 A (n 11), whereas E1537Q and E1537A generated smaller Ba2 currents (see i also Table 1).
The first argument is circumstantial and pertains to the significant kinetic differences between E1537Q and E1537A, despite similar expression levels.
Indeed, for all Vm 10 mV, whole-cell Ba2 and Ca2 currents recorded for E1537Q were not significantly different in size than E1537A currents (Fig.
4 D, the superimposed yet not normalized whole-cell Ca2 current traces measured at Vm 10 mV for the wild-type, the E1537Q, and the E1537A channels yielded similar current amplitudes when recorded under the same experimental conditions.
Yet again, E1537Q currents displayed Bernatchez et al.
E1537 Mutations in Calcium Channel Inactivation 1733 FIGURE 4 Whole-cell currents for wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, and E1537Q/ 2b / 2a channels were recorded in the presence of 10 mM Ba2 (upper panels) and 10 mM Ca2 (lower panels) by the pulse protocol previously described.
Ba2 inactivation was faster for wild-type E1537Q E1537A channels.
On average, I-V curves peaked at the following voltages: 6 2 mV (n 11) for the wild-type 1C/ 2b / 2a, 0 0.4 mV (n 15) for E1537A/ 2b / 2a, and 10 1 mV (n 14) for E1537Q/ 2b / 2a channels.
(C) Ba2 whole-cell current amplitude was higher on average for the wild-type channel with a peak current of 1.2 0.1 A (n 11) as compared to peak currents of 0.81 0.06 A (n 15) for E1537A/ 2b / 2a and 0.63 0.04 A (n 14) for a E1537Q/ 2b / 2a channels.
Current traces were not normalized because in that particular case, wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, and E1537Q/ 2b / 2a channels yielded whole-cell Ca2 currents in the same range.
E1537Q channels yielded current traces with an intermediate inactivation time course.
Wild-type and E1537Q Ca2 currents peaked, respectively, at Vm 14 1 mV (n 14) and 19 3 mV (n 11), whereas E1537A Ca2 currents peaked at Vm 5 1 mV (n 14).
(F) Whole-cell Ca2 current amplitude was higher on average for the wild-type channel with a peak current of 0.61 0.08 A (n 14) as compared to peak currents of 0.43 0.05 A (n 14) for E1537A/ 2b / 2a and 0.27 0.04 A (n 11) for E1537Q/ 2b / 2a channels.
E1537 Mutations in Calcium Channel Inactivation 1735 tigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels.
The other mutant channels showed intermediate steady-state inactivation properties with a fractional inactivation of 42 3% (n 11) for E1537Q, 39 3% (n 3) for E1537S, and 42 2% (n 4) for E1537S channels.
After a 5-s voltage pulse of 10 mV, whole-cell wild-type currents were inactivated at 83 5% (n 11), as 1 compared to 75 2% (n 4) of the E1537Q currents, 80 % (n 4) of the E1537G currents, and 81 5% (n 4) of FIGURE 6 The voltage dependence of inactivation was investigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels at the end of a 5-s prepulse.
Other mutant channels showed intermediate steady-state inactivation properties with fractional inactivation of 42 3% (n 11) for E1537Q, 39 3% (n 3) for E1537S, and 42 2% (n 4) for E1537S channels.
1): z 2.4 0.2, E0.5 20 0.7 mV (wild-type); z 1.4 0.1, E0.5 18 2 mV (E1537Q), z 1.2 0.2, E0.5 19 1 mV (E1537S); z 1.3 0.1, E0.5 17 2 mV (E1537G).
At 10 mV, whole-cell wild-type currents (F) were inactivated at 83 5% (n 11), as compared to the inactivation level of 75 2% (n 4) for the E1537Q currents (j), 80 1% (n 4) for E1537G (,), 81 5% (n 4) for E1537S ().
The fit parameters were z 2.8 0.2 and E0.5 s 22 1 mV (wild-type); z 1.9 0.2 and E0.5 18 2 mV (E1537G); z 1.9 .2 and E0.5 16 1 mV (E1537S); z 1.6 0.4 and E0.5 22 3 mV (E1537Q); z 3.0 0.3 and E0.5 17.0 0.7 mV (E1537A).
Mutant channels E1537Q, E1537S, E1537G, and E1537A were found to display significantly slower inactivation kinetics in Ba2 and Ca2 solutions.
E1537S
protein
substitution
P15381
true positive
E1537 mutant channels displayed slower inactivation kinetics in Ba2 and Ca2 solutions To investigate the possible role of the negatively charged E1537 residue in calcium-dependent inactivation, mutant channels E1537D, E1537Q, E1537S, E1537G, and E1537A were expressed in Xenopus oocytes.
2 shows typical whole-cell current recordings, for the wild-type and mutant E1537Q, E1537S, E1537A cardiac 1C calcium channels, i imax 1 1 Yo R zF Vm E0.5 exp T 1 ( 1) where i is the peak current obtained after a 5-s pulse to voltage Vm; imax is the peak current measured after a 5-s voltage pulse to 100 mV; and i/imax is their ratio when normalized to 1; Y0 is the fraction of noninactivating current; E0.5 is the midpotential of inactivation; z is the slope factor; and R, T, F have their usual meanings.
Wildtype 1C and mutant E1537Q, E1537S, E1537A were expressed in Xenopus oocytes with auxiliary 2b a nd 2a subunits.
Inactivation time con- TABLE 1 Whole-cell peak currents for wild-type 1C/ 2b / 2a, E1537A/ 2b / 2a, E1537G/ 2b / 2a, E1537Q/ 2b / 2a, and E1537S/ 2b / 2a channels in 10 mM Ba2 and 10 mM Ca2 solutions as the mean SEM of n independent experiments Wild-Type 10 Ba 10 Ca 1.3 0.61 0.2 (11) 0.08 (14) E1537A 0.87 0.43 0.06 (15) 0.05 (14) E1537G 0.38 0.25 0.06 (7) 0.03 (5) E1537Q 0.63 0.27 0.04 (24) 0.04 (11) E1537S 0.44 0.28 0.04 (8) 0.03 (7) Whole-cell currents were typically larger for the wild-type channel.
E1537 Mutations in Calcium Channel Inactivation 2a 1731 2a TABLE 2 Inactivation time constants for wild-type 1C/ 2b / 2a, E1537Q/ 2b / E1537A/ 2b / 2a channels recorded in 10 mM Ba2 or 10 mM Ca2 solutions w , E1537G/ 2b / i snact low , E1537S/ 2b / ( 2a , and Aslow Ca2 41 55 60 63 65 4% 6% 3% 5% 7% Channels 672 780 876 988 2501 B inact a (ms) f inact ast Ca2 ( ms) Afast Ca2 59 45 40 37 35 4% 6% 3% 5% 7% Ca2 ms) t 1C/ 2b / 2a E1537Q/ 2b / 2a E1537G/ 2b / 2a E1537S/ 2b / 2a E1537A/ 2b / 2a 43 (7) 26 (6)* 40 (5)** 40 (5)** 740 (5)** 49 64 69 55 56 2 (15) 10 (6)* 11 (5)* 9 (6)* 3 (12)* 496 640 701 712 2372 33 (15) 9 (6)*** 18 (5)*** 41 (6)*** 659 (12)*** Whole-cell current traces recorded at Vm 10 mV were fitted to single-exponential (Ba2 ) or double-exponential (Ca2 ) functions at time t 2 s (see Eq.
stants at Vm 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in ( the presence of 10 mM Ba2 (right panel) or 10 mM Ca2 left panel).
Ba2 inactivation time constants increased from 672 43 ms (n 7) for the wild-type 1C channel to 988 40 ms (n 5) for E1537S and to 2501 740 ms (n 5) for E1537A.
Indeed, ina ct for the Ba wild-type channel was different from the E1537Q channel time constant at the level p 0.1 (*), whereas they were significantly different at the level p 0.05 (**) for E1537G, E1537S, and E1537A channels.
On the other hand, the slower Ca2 nactivation time constant inowt increased from 496 33 ms sl ac (n 15) for the wild-type 1C channel to 712 41 ms (n 6 ) for E1537S and to 2378 660 ms (n 12) for E1537A.
Inactivation time constants for Vm sl ac 10 mV were estimated at time t 2 s for wild-type 1C/ 2b / 2a; E1537Q/ 2b / 2a, E1537G/ 2b / 2a, E1537S/ 2b / 2a, and E1537A/ 2b / 2a channels in the presence of 10 mM Ba2 (right) or 10 mM Ca2 (left).
Ba2 inactivation time constants increased from 672 43 ms (n 7) for the wild-type 1C channel to 988 40 ms (n 5) for E1537S and to 2501 740 ms (n 5) for E1537A.
On the other hand, the slower Ca nactivation time constant inowt increased from 496 33 ms (n 15) sl ac for the wild-type 1C channel to 712 41 ms (n 6) for E1537S and to 2378 660 ms (n 12) for E1537A.
ina ct for faa Ba inact E1537Q was different at the level p 0.1 (*), and Ba for E1537G, E1537S, and E1537A was different at the level p 0.05 (**).
Furthermore, inowt in Ca2 sl ac inact 2 a nd Ba in Ba ppeared remarkably similar for all calcium channel combinations and were found to increase in parallel in E1537Q, E1537S, E1537G, and E1537A channels.
E1537 Mutations in Calcium Channel Inactivation 1735 tigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels.
The other mutant channels showed intermediate steady-state inactivation properties with a fractional inactivation of 42 3% (n 11) for E1537Q, 39 3% (n 3) for E1537S, and 42 2% (n 4) for E1537S channels.
After a 5-s voltage pulse of 10 mV, whole-cell wild-type currents were inactivated at 83 5% (n 11), as 1 compared to 75 2% (n 4) of the E1537Q currents, 80 % (n 4) of the E1537G currents, and 81 5% (n 4) of FIGURE 6 The voltage dependence of inactivation was investigated for the wild-type 1C/ 2b / 2a, E1537Q/ 2b / 2a, E1537S/ 2b / 2a, E1537G/ 2b / 2a, and E1537A/ 2b / 2a channels at the end of a 5-s prepulse.
Other mutant channels showed intermediate steady-state inactivation properties with fractional inactivation of 42 3% (n 11) for E1537Q, 39 3% (n 3) for E1537S, and 42 2% (n 4) for E1537S channels.
1): z 2.4 0.2, E0.5 20 0.7 mV (wild-type); z 1.4 0.1, E0.5 18 2 mV (E1537Q), z 1.2 0.2, E0.5 19 1 mV (E1537S); z 1.3 0.1, E0.5 17 2 mV (E1537G).
At 10 mV, whole-cell wild-type currents (F) were inactivated at 83 5% (n 11), as compared to the inactivation level of 75 2% (n 4) for the E1537Q currents (j), 80 1% (n 4) for E1537G (,), 81 5% (n 4) for E1537S ().
The fit parameters were z 2.8 0.2 and E0.5 s 22 1 mV (wild-type); z 1.9 0.2 and E0.5 18 2 mV (E1537G); z 1.9 .2 and E0.5 16 1 mV (E1537S); z 1.6 0.4 and E0.5 22 3 mV (E1537Q); z 3.0 0.3 and E0.5 17.0 0.7 mV (E1537A).
736 Biophysical Journal Volume 75 October 1998 the E1537S currents.
Mutant channels E1537Q, E1537S, E1537G, and E1537A were found to display significantly slower inactivation kinetics in Ba2 and Ca2 solutions.
D1535A
protein
substitution
P15381
true positive
Our observations that the E1537 mutations failed to abolish calciumdependent inactivation but yielded slower inactivating channels agree with the study of the 1C triple mutant D1535A Bernatchez et al.
The triple mutant failed to abolish calcium-dependent inactivation, although the Ca2 current traces they recorded from the 1C triple mutant (D1535A E1537A D1546A) were actually twice as slow as the ones recorded for the wild-type channel.
11090098
full text
H404T
protein
substitution
P16390
true positive
5 Since open block kinetics were shown to be similar for wild type mKv1.3 and the H404T mutant mKv1.3 channel, and since the block of the H404T mutant channels by verapamil could be described exactly by a simple three-state open block model, the mutant channel could serve as a screening channel to determine open block anities of new PAA derivatives in high through-put experiments.
In addition, experiments with double mutants (H404T+XnnnY) could now be envisioned trying to identify the verapamil binding site at the channel.
Expression The mKv1.3 wild type gene and the mKv1.3 H404T mutant gene (a generous gift from Dr K.
To recreate the current decay of inactivation and block and to check the predictions of the model, we simulated the channel states according to scheme I (wild type) or II (H404T mutant) shown in Figure 1, so that state O overlays the original normalized current.
(v) l(40 mV) was manually adjusted during simulation (wild type) or deduced from the steady-state currents during depolarization (H404T mutant), according to: l=1/(R*tcorr).
In the H404T mutant channels h was set to 0 in the simulations.
In the H404T mutant channels h* was set to 0 for the simulations.
Using high [K+]o as the bathing solution dierences in the verapamil block characteristics can be observed compared to B r i t i s h Jo u r n a l o f P h a r m a c o l o g y v o l 1 3 1 ( 7 ) Figure 1 Simplied scheme of Kv1.3 transitions by depolarization in the absence and presence of verapamil with (scheme I, for wild type mKv1.3 channels) and without (scheme II, for H404T mutant mKv1.3 channels) C-type inactivation.
3 i n a c ti v a t i o n a n d a c c u m u l a t i o n o f v e r a p a m i l b l o c k Figure 2 Basic properties of verapamil to block current through wild type and mutant H404T mKv1.3.
To see if there is a link between inactivation, recovery from inactivation and accumulation of verapamil block we tested verapamil on the H404T mutant Kv1.3 channel.
The current decay of the H404T mutant channel during depolarization in the presence of verapamil, shown in Figure 2E, was qualitatively similar to wild type; however, steady-state currents at the end of the depolarization were clearly detectable.
In addition, in the H404T mutant no further reduction of peak currents beyond the rst pulse after verapamil wash-in was observed (see Figure 2F).
After that, no accumulation of block occurred in the H404T mutant channel, even if we increased the pulse rate from 2 min71, as used for the accumulation of block in wild type and in Figure 2B,D,F, to 30 min71 (data not shown).
Figure 3A,C and E show superimposed tail currents elicited by depolarizing test pulses with varying durations for the wild type under control (high [K+]o) conditions (Figure 3A) and with verapamil (Figure 3C), and for the mutant H404T under similar (high [K+]o) conditions with verapamil (Figure 3E).
E, tail currents elicited in the H404T mutant in high [K+]o and 50 mM verapamil were independent of the applied duration of depolarization, here 200 and 1250 ms, respectively.
In contrast to wild type, the tail currents of the mutant H404T channels were independent of the duration of the depolarizing pulse (Figure 3E and F).
The dotted line in Figure 5B represents a mono-exponential t to the data with 736 mV per e-fold for the wild type mKv1.3 channel (in the H404T mutant channels a voltage dependence R.J.
Current simulation To recreate the current decay during development of block and to obtain the time courses of the other states, we performed a simulation based on the scheme I and II (Figure 1) for wild type and the H404T mutant channels, respectively.
7.5+1.1 (n=5) 7.1+2.0 (n=10) 8.2+1.4 (n=9) 2.8 4.8 220+91 (n=42) 620+250 (n=12) *30+14 (n=10) 230+91 (n=8) 15+4.9 (n=18) 10+3.1 (n=6) 78+16 (n=7) 15+3.3 (n=5) H404T mutant mKv1.3 high [Na+]o high [K+]o 1.8+0.47 * 106 (n=15) 1.5+0.27 * 106 (n=10) n.d.
3 i n a c ti v a t i o n a n d a c c u m u l a t i o n o f v e r a p a m i l b l o c k The H404T mutant mKv1.3 channel To do so, we used the H404T mutant mKv1.3 channel known for its slow inactivation (Aiyar et al., 1995; Rauer & Grissmer, 1996).
The dierent blocking behaviors of wild type and H404T mutant mKv1.3 channel by the phenylalkylamine verapamil could be attributed to comparable open block properties, as the onand o-rate constants were similar in both channels.
The apparent dierences in block (steady-state, tail-integrals, recovery, accumulation) of both channels were caused by the absence of intrinsic C-type inactivation in the H404T mutant channel (compare scheme I and II, Figure 1).
H404T mutant: (C) 50 mM verapamil in high [K+]o according to scheme II of Figure 1), as expected for a depolarizing pulse to 40 mV from a holding potential, that has allowed complete recovery from block.
Figure 6c shows the simulation of the H404T mutant Kv1.3 channels according to scheme II (Figure 1).
In addition, in order to produce new PAA-derivatives, which are Kv1.3-selective over L-type Ca2+ channels, the H404T mutant mKv1.3 channels could be used to search for openchannel blocking drugs in high through-put screens.
10097181
full text
R615L
protein
substitution
true positive
P11716
C a f f e i n e - i n d u c e d C a2 r e l e a s e a n d t h a p s i g a r g i n - i n d u c e d Ca2 release in single transfected HEK-293 cells Caf feine-induced Ca2 release, nM 874 517 212 609 578 249 68 50** 41** 27* 50* 39** Thapsigargin-induced Ca2 release, nM ( n ) 348 273 120 320 236 110 28 (27) 27 (19)* 34 (12)** 31 (12) 41 (10)* 21 (17)** Constr uct RyR1 R615L Y523S RyR1 RyR1 RyR1 RyR1 R615L Y523S I4897T K d[Ca2 ], Ca2 c oncentration required for half-max imal r yanodine binding.
I4898T
protein
substitution
true positive
P21817
In a lar ge Mex ican k indred w ith an unusually severe and highly penet rant for m of the disorder, DNA sequencing identif ied an I4898T mut ation in the Cter minal t ransmembrane luminal reg ion of the RyR1 pr otein that c onstitutes the skelet al muscle r yanodine receptor.
The I4898T mut ation was int r oduced into a rabbit RYR1 cDNA and ex pressed in HEK-293 cells.
Compar ison w ith t wo other c oex pressed mut ant normal channels suggests that the I4898T mut ation pr oduces one of the most abnor mal RyR1 channels yet investigated, and this level of abnor malit y is ref lected in the severe and penet rant phenot ype of af fected cent ral c ore disease individuals.
Ex pression and Functional A nalysis of I4898T Mut ant.
Pedigree of the Mex ican family show ing: CCD st atus, MHS st atus, haplot ypes c onstr ucted w ith markers for loci D19S220 and D19S47, and the presence of the I4898T mut ation.
The presence or absence of the I4898T mut ation is indicated by or , respectively.
Sequence and detection of the I4898T mut ation.
Af fected indiv iduals in the CCD pedig ree are heteroz ygous for the I4898T mut ation.
By c ontrast, I4898T represents a novel t ype of MHS mut ation as it resides in the C t e r m i n u s o f t h e R y R 1 p r o t e i n t h a t f o r m s t h e C a2 c h a n n e l (27).
Therefore, perturbation of the put ative triadin binding site by the I4898T mut ation may disr upt molecular signaling bet ween RyR1 and calsequestrin resulting in abnor mal RyR1 channel gating.
The I4898T mut ation is the first RYR1 mut ation to be associated w ith a highly penetrant and severe for m of CCD in a large family.
Cellular Ca2 photometr y and imaging assays indicate that the I4898T mut ation produces the most abnor mal diseaseassociated RyR1 channel yet investigated, mirroring the severe CCD phenot ype associated w ith the mut ation.
I4897T
protein
substitution
true positive
P11716
Const r uction of the I4897T Mut ation in Rabbit RYR1 cDNA.
The I4897T mut ation was introduced into RyR1 cassette 11 (pCS11) by using the QuickChange site-directed mut agenesis protoc ol (Strategene).
The ClaI HindIII f ragment of pCS11-I4897T then was exchanged for the same region in full-length pcDNA-RyR1.
By c ontrast, the I4897T mut ant channel exhibited little or no caf feine-induced Ca2 release in the assay (Fig.
Coex pression of SERCA1 w ith the I4897T r mut ant, however, did not re-est ablish caf feine-induced Ca2 elease (Fig.
Results of Ca2 photometr y and imaging assays and ex pression of the nor mal and I4897T mut ant RYR1 cDNAs in HEK-293 cells.
Traces of the responses in the Ca2 photometr y assay to increment al doses of caf feine in HEK-293 cells ex pressing: ( A ) the pcDNA3 vector, ( B ) nor mal RyR1, ( C ) I4897T mut ant, ( D ) RyR1 plus SERCA1, ( E ) I4897T plus SERCA1, and ( F ) I4897T plus RyR1 plus SERCA1.
The Ca2 imaging assay demonstrates caf feine-induced Ca2 release in a single HEK-293 cell ex pressing: ( G ) RyR1 plus SERCA1 or ( H ) I4897T plus RyR1 plus SERCA1.
( I ) Western blot of HEK-293 cell extracts 48 hr af ter transfection w ith pcDNA3 vector, nor mal RyR1, and I4897T mut ant c onstr ucts.
C o m p a r i s o n o f c a f f e i n e - a n d h a l o t h a n e - i n d u c e d C a2 and I4897T mut ant r yR1 receptors Caf feine-induced Ca2 Constr uct RyR1 I4897T RyR1 SERCA1 I4897T SERCA1 I4897T S RyR1 ERCA1 r release P roc.
A nalysis of luminal Ca2 c oncentration by using this approach showed a sign ificant decrease in the size of luminal Ca2 stores in cells c otransfected w ith nor mal RYR1 and I4897T cDNAs (Table 4).
This result suggests that the decreased agon istinduced Ca2 release, which was obser ved in the heteroz ygous I4897T cells, was caused by a reduced luminal Ca 2 c oncentration resulting f rom a leaky mut ant channel.
Further more, the cells ex pressing the I4897T mut ant channels had the lowest thapsigargin-induced Ca2 release (Table 4).
C o m p a r i s o n o f [3H ] r y a n o d i n e b i n d i n g f o r n o r m a l a n d I4897T mut ant RyR1 receptors Constr uct RyR1 I4897T I4897T B max, fmol mg protein (n) 73.1 3.0 11.3 3.0 (3) 1.7* (4) 1.1* (3) K d[Ca2 ], nM (n) 270 69 30 (3) 24* (3) philia and is maint ained in all k nown cardiac ( RYR2) and brain (RYR3) isofor ms.
The nor mal caf feine and halothane sensitiv ities obser ved in the I4897T normal heteroz ygous mut ant cells were unexpected, because the t wo patients investigated by the I VCT had increased sensitiv it y to caf feine- and halothane-induced muscle c ontractures.
Unlike prev iously characterized RyR1 mut ations (33, 34), the I4897T mut ation reduces [3H]r yanodine binding, possibly by perturbing the ligand binding site that is located in the C d t e r m i n u s o f t h e p r o t e i n ( 2 5 ) .
H o w e v e r a n a l y s i s o f t h e C a2 ependence of r yanodine binding in the I4897T normal heteroz ygous cell lysates demonstrated that the mut ant channel is half-activated by a 4-fold lower Ca 2 c oncentration than the nor mal channel.
C a f f e i n e - i n d u c e d C a2 r e l e a s e a n d t h a p s i g a r g i n - i n d u c e d Ca2 release in single transfected HEK-293 cells Caf feine-induced Ca2 release, nM 874 517 212 609 578 249 68 50** 41** 27* 50* 39** Thapsigargin-induced Ca2 release, nM ( n ) 348 273 120 320 236 110 28 (27) 27 (19)* 34 (12)** 31 (12) 41 (10)* 21 (17)** Constr uct RyR1 R615L Y523S RyR1 RyR1 RyR1 RyR1 R615L Y523S I4897T K d[Ca2 ], Ca2 c oncentration required for half-max imal r yanodine binding.
The obser ved lower luminal Ca2 ontent and elevated c y toplasmic Ca2 c oncentrations c ould result f rom this hypersensitiv it y to Ca2 as the I4897T mut ant channel is activated at the resting cellular Ca2 c oncentration while the nor mal RyR1 channel remains closed.
Y523S
protein
substitution
true positive
P11716
C a f f e i n e - i n d u c e d C a2 r e l e a s e a n d t h a p s i g a r g i n - i n d u c e d Ca2 release in single transfected HEK-293 cells Caf feine-induced Ca2 release, nM 874 517 212 609 578 249 68 50** 41** 27* 50* 39** Thapsigargin-induced Ca2 release, nM ( n ) 348 273 120 320 236 110 28 (27) 27 (19)* 34 (12)** 31 (12) 41 (10)* 21 (17)** Constr uct RyR1 R615L Y523S RyR1 RyR1 RyR1 RyR1 R615L Y523S I4897T K d[Ca2 ], Ca2 c oncentration required for half-max imal r yanodine binding.
12761351
full text
L1280A
protein
substitution
true positive
P15390
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
In resting channels, the blocking potency of S( )-bupivacaine was slightly increased in L1280A, L1280T, and L1280D.
The blocking potency of R( )-bupivacaine was selectively decreased in mutants L1280A, L1280N, L1280Q, L1280D, and L1280E.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
It is interesting that mutation L1465A in the rat brain Nav1.2 channel, which corresponds to L1280A in the rat skeletal muscle Nav1.4 channel, showed significantly reduced affinity of inactivated channels for the LA etidocaine in electrophysiological experiments in the oocyte expression system (Yarov-Yarovoy et al., 2001).
In our experiments, block of resting and inactivated channels by bupivacaine enantiomers in mutation L1280A was only slightly changed compared with wild-type channels.
L1280E
protein
substitution
true positive
P15390
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
At this potential, block of L1280E, L1280N, L1280Q, and L1280R did not yet reach a plateau, so we might have slightly underestimated the block of inactivated channels in these mutants.
Blocking potency of R( )-bupivacaine was slightly decreased in L1280E, L1280K, and L1280R.
The blocking potency of R( )-bupivacaine was selectively decreased in mutants L1280A, L1280N, L1280Q, L1280D, and L1280E.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
Mutation L1280E, carrying another negatively charged but larger residue, revealed a similar pattern.
L1280D
protein
substitution
true positive
P15390
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
In all mutant channels, the midpoint voltages of activation were significantly shifted rightward by approximately 6 to 22 mV compared with wild-type, except for mutant L1280D, in which the rightward shift was not statistically significant.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
In resting channels, the blocking potency of S( )-bupivacaine was slightly increased in L1280A, L1280T, and L1280D.
The blocking potency of R( )-bupivacaine was selectively decreased in mutants L1280A, L1280N, L1280Q, L1280D, and L1280E.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
However, in contrast to mutation N434D, bupivacaine block of inactivated channels was not increased in mutation L1280D but was unchanged for S( )-bupivacaine and slightly decreased for R( )-bupivacaine.
L1280F
protein
substitution
true positive
P15390
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
Unfortunately, two other mutations of that type, L1280F and L1280Y, did not express sufficient current for further analysis.
L1280C
protein
substitution
true positive
P15390
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
Current densities for sufficiently expressing mutants all were significantly smaller compared with wild-type except for mutants L1280C and L1280N (Table 1).
The steepness of the activation curves were significantly decreased in all mutants compared with wild-type channels except for mutants L1280C and L1280K, in which the decrease was not statistically significant (Table 1).
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
L1280K
protein
substitution
true positive
P15390
Block of inactivated channels was increased in a mutation containing an aromatic group (L1280W) and decreased in mutations containing a positive charge (L1280K, L1280R).
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
The steepness of the activation curves were significantly decreased in all mutants compared with wild-type channels except for mutants L1280C and L1280K, in which the decrease was not statistically significant (Table 1).
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Block of inactivated channels by R( )- or S( )bupivacaine was slightly increased in mutant L1280W and was dramatically decreased in mutant L1280K.
Blocking potency of R( )-bupivacaine was slightly decreased in L1280E, L1280K, and L1280R.
The blocking potency of both enantiomers was decreased in mutants L1280K and L1280R.
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
Bupivacaine block of inactivated channels was likewise decreased in mutations L1280K and L1280R carrying a positively charged residue.
N434D
protein
substitution
true positive
P15390
However, in contrast to mutation N434D, bupivacaine block of inactivated channels was not increased in mutation L1280D but was unchanged for S( )-bupivacaine and slightly decreased for R( )-bupivacaine.
N434E
protein
substitution
true positive
P15390
Effects of Mutations N434E and N434Q on Activation, Steady-State Inactivation, and State-Dependent Block by Bupivacaine Enantiomers.
To test whether glutamate and glutamine are likewise able to cause bupivacaine stereoselectivity while residing in position N434, we created mutations N434E and N434Q and studied their gating properties and state-dependent interaction with bupivacaine enantiomers.
Activation and inactivation properties of mutations N434E and N434Q were characterized with standard pulse protocols as described in Fig.
Original current traces of N434E and N434Q recorded to estimate activation properties are shown in Fig.
Mutation N434E exhibited rectification at positive voltages and significant sustained currents at the end of a 5-ms depolarization (Fig.
The midpoint voltage of steady-state inactivation was unchanged in mutant N434E and was shifted rightward by 10 mV in mutant N434Q compared with wild-type channels (Fig.
The effects of 10 M R( )- and S( )-bupivacaine on mutants N434E and N434Q elicited after prepulses to 140 and 70 mV are shown in Fig.
The normalized peak currents of mutant N434E and N434Q in control and in the presence of 10 M R( )-bupivacaine and S( )-bupivacaine as a function of different conditioning prepulse potentials are shown in Fig.
Block of resting channels by R( )or S( )-bupivacaine was decreased in mutant N434E and was unchanged in mutant N434Q.
In mutant N434E, block of inactivated channels by S( )-bupivacaine was selectively decreased, resulting in a significant stereoselectivity.
The incomplete inactivation from the open state observed in mutation N434E is consistent with the proposed importance of residues at the intracellular end of segment D1-S6, among others, for closure of the fast inactivation gate (Yarov-Yarovoy et al., 2002).
The peculiar rectification at positive voltages in mutation N434E (Fig.
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
5.Activation and steady-state inactivation of mutants Nav1.4-N434E and Nav1.4-N434Q.
Na currents through mutants Nav1.4-N434E (A) and Nav1.4-N434Q (B) were evoked by 5-ms pulses ranging from 120 to 50 mV from a holding potential of Vh 140 mV.
C and D, data for activation and steady-state inactivation of mutants Nav1.4-N434E and Nav1.4-N434Q were obtained as described in Fig.
State-dependent block of Nav1.4-N434E and Nav1.4-N434Q by bupivacaine enantiomers.
C and D, data of normalized Nav1.4-N434E and Nav1.4-N434Q currents in control channels (E) and with 10 M R( )- (f) and S( )-bupivacaine (}), plotted as a function of the conditioning prepulse potential, were obtained as described in Fig.
A comparable result was obtained for mutation N434E, in which block by S( )-bupivacaine was selectively decreased, resulting in a moderate bupivacaine stereoselectivity.
L1280N
protein
substitution
true positive
P15390
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
Current densities for sufficiently expressing mutants all were significantly smaller compared with wild-type except for mutants L1280C and L1280N (Table 1).
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
At this potential, block of L1280E, L1280N, L1280Q, and L1280R did not yet reach a plateau, so we might have slightly underestimated the block of inactivated channels in these mutants.
The blocking potency of R( )-bupivacaine was selectively decreased in mutants L1280A, L1280N, L1280Q, L1280D, and L1280E.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
L1280Q
protein
substitution
true positive
P15390
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
At this potential, block of L1280E, L1280N, L1280Q, and L1280R did not yet reach a plateau, so we might have slightly underestimated the block of inactivated channels in these mutants.
The blocking potency of R( )-bupivacaine was selectively decreased in mutants L1280A, L1280N, L1280Q, L1280D, and L1280E.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
L1280R
protein
substitution
true positive
P15390
Block of inactivated channels was increased in a mutation containing an aromatic group (L1280W) and decreased in mutations containing a positive charge (L1280K, L1280R).
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels.
At this potential, block of L1280E, L1280N, L1280Q, and L1280R did not yet reach a plateau, so we might have slightly underestimated the block of inactivated channels in these mutants.
Blocking potency of R( )-bupivacaine was slightly decreased in L1280E, L1280K, and L1280R.
The blocking potency of both enantiomers was decreased in mutants L1280K and L1280R.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
Bupivacaine block of inactivated channels was likewise decreased in mutations L1280K and L1280R carrying a positively charged residue.
The amount of stereoselectivity [R( )/S( )] for L1280R, Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1405 responsible for the flexibility of other S6 segments in the channel opening (Jiang et al., 2002).
L1280T
protein
substitution
true positive
P15390
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
In resting channels, the blocking potency of S( )-bupivacaine was slightly increased in L1280A, L1280T, and L1280D.
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
L1280W
protein
substitution
true positive
P15390
Block of inactivated channels was increased in a mutation containing an aromatic group (L1280W) and decreased in mutations containing a positive charge (L1280K, L1280R).
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Block of inactivated channels by R( )- or S( )bupivacaine was slightly increased in mutant L1280W and was dramatically decreased in mutant L1280K.
In inactivated channels, the blocking potency of bupivacaine enantiomers was increased only in mutant L1280W.
Significant stereoselectivity was revealed in mutants L1280W, L1280A, L1280N, L1280Q, L1280D, L1280E, and L1280R and was greatest in mutants L1280R [R( )/S( ) 7], L1280E [R( )/S( ) 5], L1280Q [R( )/S( ) 4], and L1280N [R( )/S( ) 2].
mutation L1280W, stereoselectivity resulted from a selective decrease in block of inactivated channels by R( )-bupivacaine.
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
Bupivacaine block of inactivated channels was likewise enhanced in mutation L1280W carrying an aromatic residue.
L1465A
protein
substitution
true positive
P04775
Mutation L1465A in Nav1.2 channels (homologous to L1280 in Nav1.4 channels), causing a positive shift, was reasoned to belong to a group of mostly hydrophobic residues on one side of the -helical segment D3-S6 that faces the lumen of the pore in the activated and inactivated state and interacts with surrounding transmembrane segments in D3 in the resting state (Yarov-Yarovoy et al., 2001).
It is interesting that mutation L1465A in the rat brain Nav1.2 channel, which corresponds to L1280A in the rat skeletal muscle Nav1.4 channel, showed significantly reduced affinity of inactivated channels for the LA etidocaine in electrophysiological experiments in the oocyte expression system (Yarov-Yarovoy et al., 2001).
L1280Y
protein
substitution
true positive
P15390
Mutants L1280A, L1280C, L1280D, L1280E, L1280K, L1280N, L1280Q, L1280R, L1280T, and L1280W expressed sufficient Na currents for further analysis, whereas mutants L1280F and L1280Y expressed little or no Na currents.
Unfortunately, two other mutations of that type, L1280F and L1280Y, did not express sufficient current for further analysis.
N434Q
protein
substitution
true positive
P15390
5 kh Current Density WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N343E N434Q 34.7 (n 16.4 (n 24.9 (n 27.9 (n 19.5 (n 20.4 (n 6.5 (n 15.5 (n 13.0 (n 20.7 (n 28.8 (n 43.7 (n 36.6 (n 1.7 7) 2.1 5) 0.8 7) 3.1 7) 0.9 5) 2.2 7) 1.4 7) 2.6 8) 3.9 7) 1.3 8) 1.2 8) 1.2 6) 1.6 7) 6.8 (n 9.9 (n 8.4 (n 11.7 (n 10.1 (n 8.3 (n 12.8 (n 11.6 (n 14.5 (n 10.0 (n 9.1 (n 7.4 (n 8.5 (n 0.7 7) 0.5 5) 0.4 7) 0.6 7) 0.6 5) 0.5 7) 0.8 7) 1.3 8) 1.4 7) 0.8 8) 0.6 8) 0.4 6) 0.3 7) mV 76.9 (n 83.8 (n 86.3 (n 84.2 (n 90.5 (n 88.4 (n 87.4 (n 83.6 (n 93.1 (n 88.4 (n 86.1 (n 72.6 (n 67.0 (n 2.0 8) 1.2 6) 1.7 6) 1.3 9) 1.8 5) 2.1 7) 2.0 5) 1.9 6) 1.2 6) 1.3 7) 1.7 8) 0.9 5) 1.8 6) 5.7 (n 6.0 (n 5.8 (n 7.5 (n 6.8 (n 6.5 (n 6.6 (n 6.1 (n 7.4 (n 6.5 (n 6.0 (n 4.9 (n 6.3 (n 0.2 8) 0.2 6) 0.3 6) 0.4 9) 0.2 5) 0.3 7) 0.3 5) 0.2 6) 0.3 6) 0.3 7) 0.4 8) 0.3 5) 0.4 6) pA/pF 253 74 210 40 50 99 174 60 50 127 109 108 328 51 10 73 10 11 17 35 11 12 29 24 26 67 Nav1.4-L1280 Mutant Channel Block by Bupivacaine Enantiomers 1401 grees of slow inactivation after prepulses 110 mV.
Effects of Mutations N434E and N434Q on Activation, Steady-State Inactivation, and State-Dependent Block by Bupivacaine Enantiomers.
To test whether glutamate and glutamine are likewise able to cause bupivacaine stereoselectivity while residing in position N434, we created mutations N434E and N434Q and studied their gating properties and state-dependent interaction with bupivacaine enantiomers.
Activation and inactivation properties of mutations N434E and N434Q were characterized with standard pulse protocols as described in Fig.
Original current traces of N434E and N434Q recorded to estimate activation properties are shown in Fig.
Activation properties of N434Q were unchanged compared with wild-type channels.
The midpoint voltage of steady-state inactivation was unchanged in mutant N434E and was shifted rightward by 10 mV in mutant N434Q compared with wild-type channels (Fig.
The effects of 10 M R( )- and S( )-bupivacaine on mutants N434E and N434Q elicited after prepulses to 140 and 70 mV are shown in Fig.
The normalized peak currents of mutant N434E and N434Q in control and in the presence of 10 M R( )-bupivacaine and S( )-bupivacaine as a function of different conditioning prepulse potentials are shown in Fig.
Block of resting channels by R( )or S( )-bupivacaine was decreased in mutant N434E and was unchanged in mutant N434Q.
In mutant N434Q, block of inactivated channels was decreased for both bupivacaine enantiomers compared with wild-type channels.
IC50 Resting R( ) S( ) R( )/S( ) R( ) IC50 Inactivated S( ) R( )/S( ) M M WT L1280A L1280C L1280D L1280E L1280K L1280N L1280Q L1280R L1280T L1280W N434E N434Q 96 (n 89 (n 84 (n 88 (n 117 (n 155 (n 96 (n 77 (n 153 (n 81 (n 73 (n 189 (n 94 (n 7 8) 6 5) 5 5) 4 4) 3 5) 9 5) 10 5) 5 5) 11 5) 3 6) 8 5) 8 5) 8 5) 98 (n 56 (n 83 (n 63 (n 97 (n 121 (n 103 (n 80 (n 125 (n 77 (n 87 (n 263 (n 120 (n 7 8) 3 7) 6 5) 5 5) 8 5) 10 5) 9 5) 6 5) 11 5) 6 7) 9 5) 10 5) 8 5) 1.0 1.6 1.0 1.4 1.2 1.3 0.9 1.0 1.2 1.1 0.8 0.7 0.8 3.8 (n 5.6 (n 4.3 (n 5.6 (n 25 (n 99 (n 7.4 (n 15 (n 58 (n 7.7 (n 1.4 (n 3.1 (n 12 (n 0.4 8) 0.6 5) 0.4 5) 0.6 4) 2 5) 10 5) 0.9 5) 1 5) 6 5) 0.3 5) 0.1 5) 0.3 5) 2 5) 4.4 (n 3.6 (n 4.8 (n 3.7 (n 4.8 (n 77 (n 3.9 (n 3.8 (n 8.1 (n 5.7 (n 2.8 (n 7.5 (n 12 (n 0.5 8) 0.6 6) 0.5 5) 0.4 5) 0.7 5) 8 5) 0.4 5) 0.5 5) 1.1 5) 0.8 6) 0.3 5) 1.1 5) 2 5) 0.9 1.6 0.9 1.5 5.2 1.3 1.9 3.9 7.2 1.4 0.5 0.4 1.0 1404 Nau et al.
5.Activation and steady-state inactivation of mutants Nav1.4-N434E and Nav1.4-N434Q.
Na currents through mutants Nav1.4-N434E (A) and Nav1.4-N434Q (B) were evoked by 5-ms pulses ranging from 120 to 50 mV from a holding potential of Vh 140 mV.
C and D, data for activation and steady-state inactivation of mutants Nav1.4-N434E and Nav1.4-N434Q were obtained as described in Fig.
State-dependent block of Nav1.4-N434E and Nav1.4-N434Q by bupivacaine enantiomers.
C and D, data of normalized Nav1.4-N434E and Nav1.4-N434Q currents in control channels (E) and with 10 M R( )- (f) and S( )-bupivacaine (}), plotted as a function of the conditioning prepulse potential, were obtained as described in Fig.
N434R
protein
substitution
true positive
P15390
Furthermore, mutation N434R exhibited significant stereoselectivity for block of inactivated channels that resulted from a selective decrease in block by S( )-bupivacaine.
More surprisingly, stereoselectivity resulted from a selective decrease in block by R( )-bupivacaine, in contrast to mutation N434R in D1-S6.
Supporting this hypothesis was the finding that mutation N434R exhibited significant stereoselectivity for the block of inactivated channels that resulted from a selective decrease in block by S( )-bupivacaine (Nau et al., 1999).
This is in contrast to mutation N434R of D1-S6, in which stereoselectivity for block of inactivated channels resulted from a selective decrease in block by S( )-bupivacaine.
As mentioned previously, this is in contrast to the findings in mutation N434R of D1-S6, in which stereoselectivity for block of inactivated channels resulted from a selective decrease in block by S( )-bupivacaine (Nau et al., 1999).
In mutant N434R, the charge, size, and orientation of atoms and bonds in the guanidinium group of arginine were held responsible for bupivacaine stereoselectivity.
12367613
full text
H101R
protein
substitution
true negative
In order to assess the consequences of this pharmacological switch in vivo, the motor and proconvulsant effects of Ro 15-4513 were analyzed in knock-in mice containing point-mutated 1(H101R)-GABAA receptors.
Furthermore the influence of the 1(H101R) substitution on the efficacy of the carboline inverse agonist DMCM was examined both in vitro and in vivo.
In 1(H101R) mice, Ro 15-4513 decreased locomotion and, at a higher dose (30 mg/kg) it displayed an anticonvulsant action.
In vitro, DMCM acted as an inverse agonist at recombinant 122 receptors whereas it potentiated GABA-evoked chloride currents at 1(H101R)22 receptors.
DMCM was inactive as a convulsant in 1(H101R) mice.
A replacement of the histidine residue 101 by an arginine in the 1 subunit (1-H101R) switches the efficacy of Ro 15-4513 from partial inverse agonism to partial agonism (Benson et al., 1998).
A histidine-to-arginine point mutation at residue 101 (H101R) in the GABAA receptor 1-subunits has been introduced into the germline of mice by gene targeting (Rudolph et al., 1999).
As expected from studies on recombinant receptors, this mutation rendered the respective receptors diazepam-insensitive and switched the efficacy of Ro 15-4513 from inverse agonism to agonism as demonstrated by the potentiation of GABAinduced chloride currents of cerebellar Purkinje cells from 1(H101R) mice (Rudolph et al., 1999).
To determine the function of the 1-GABAA receptors with regard to the pharmacological effects of benzodiazepine site inverse agonists, we firstly focused on the analysis of the motor and proconvulsant effects of Ro 15-4513 in 1(H101R) mice.
Then, we examined the influence of the 1(H101R) point mutation on the pharmacological efficacy of the -carboline full inverse agonist DMCM both in vitro and in vivo.
The modulation of GABA-evoked chloride currents by DMCM was studied electrophysiologically in transfected HEK 293 cells expressing wild type 122 or 1(H101R)22 GABAA receptors.
The convulsant action of DMCM was tested in wild type and 1(H101R) mice.
Electrophysiology Cultured human embryonic kidney (HEK) cells 293 were transiently transfected with rat cDNA coding for wild type or H101R point mutated 1(H101R)-subunits in combination with 2 and 2-subunits as described in Benson et al.
Subjects Female 1(H101R) mice bred on the 129/SvEv background (3rd backcross), 1(H101R) mice bred on the 129/SvJ background (6th to 9th backcross to the 129/SvJ background) and the respective wild type control mice were used (Rudolph et al., 1999).
Effects of Ro 15-4513 in the proconvulsant pentylenetetrazole test in wild type and a1(H101R) mice In wild type mice, a pretreatment with Ro 15-4513 (10 mg/kg per os) followed by 50 mg/kg of pentylenetetrazole reduced the latency to the first myoclonic jerk episode (p 0.01 in comparison to vehicle p entylenetetrazole) (Fig.
This proconvulsant effect was absent in 1(H101R) mice since the latency to convulsions was similar in mutant mice which were pretreated with either vehicle or Ro 15-4513 [Genotype X Treatment F(1,36) 9.48, p 0.01].
Wild type and 1(H101R) mice pretreated with vehicle showed a similar heightened latency to the first myoclonic jerk episode following the pentylenetetrazole injection.
To exclude any alteration in pentylenetetrazole sensitivity as a potential explanation for the absence of proconvulsant action of Ro 15-4513 in 1(H101R) mice, a second series of mice were pretreated with either vehicle or 10 mg/kg of Ro 15-4513 and 15 min later they were injected with a higher dose (60 mg/kg) of pentylenetetrazole.
Under these conditions, mice pretreated with vehicle developed tonic convulsions with a similar median latency in wild type [143 s (70600, range)] and 1(H101R) mice [104 s (53-600)] (n 5 per group).
In contrast, whereas a pretreatment with Ro 15-4513 shortened significantly the latency to tonic convulsions in wild type mice [52 s (34 85); p 0.05 versus vehicle], it was inactive in 1(H101R) mice [112 s (82129)] (n 5 per group).
Effects of Ro 15-4513 on baseline locomotion in wild type and a1(H101R) mice In wild type mice, a treatment with Ro 15-4513 at the dose of 10 mg/kg produced an increase in baseline locomotor activity in a familiar context (p 0.05 compared to vehicle) (Fig.
Ro 15-4513 displayed an opposite motor effect in 1(H101R) mice as evidenced by the drug-induced significant diminution of the baseline level of locomotion in comparison to vehicle treatment [Genotype X Treatment, F(1,14) 16.71, p 0 .001].
Wild type and 1(H101R) mice treated with vehicle displayed similar baseline locomotion.
Effects of Ro 15-4513 on baseline locomotion in wild type and 1(H101R) mice.
Effects of Ro 15-4513 in the proconvulsant pentylenetetrazole test in wild type and 1(H101R) mice.
/ Neuropharmacology 43 (2002) 679684 tylenetetrazole in both wild type and 1(H101R) mice (Fig.
2) may have masked a potential switch in efficacy of Ro 15-4513 from a proconvulsant to an anticonvulsant action in 1(H101R) mice.
Therefore, in a separate experiment, the effects of a higher dose (30 mg/kg) of Ro 15-4513 in combination with 50 mg/kg of pentylenetetrazole was studied in 1(H101R) mice bred on the 129/SvJ background.
Following the pentylenetetrazole injection, 1(H101R) mice pretreated with 30 mg/kg of Ro 15-4513 developed the first myoclonic jerk episode at a significantly higher latency than mutant mice pretreated with vehicle (p 0.05), thus revealing an anticonvulsant action of Ro 15-4513 in the 1(H101R) mice (Fig.
Effect of DMCM on GABA-evoked chloride currents for wild type and a1(H101R)-GABAA receptors expressed in transfected HEK 293 cells DMCM applied at the concentration of 1 M decreased the amplitude of GABA-evoked chloride currents in cells expressing wild type 122 receptors (Fig.
However, in cells expressing 1(H101R)22 receptors, the same treatment with DMCM produced an opposite effect as shown by the percent enhancement of the GABA-evoked chloride currents.
Thus, in the same way as for Ro 15-4513, the H101R point mutation in the 1-subunit switched the mode of action of DMCM from inverse agonism to agonism in vitro.
(A) Effects of DMCM on GABA-evoked chloride currents for 122 and 1(H101R)22 GABAA receptors expressed in HEK 293 cells.
(B) Single traces of the effects of DMCM on the GABA-evoked chloride currents in HEK 293 cells expressing 122 or 1(H101R)22 receptors.
Anticonvulsant effect of Ro 15-4513 (30 mg/kg per os) in 1(H101R) mice.
Effects of DMCM in wild type and a1(H101R) mice To assess in vivo the consequences of the H101R point mutation-induced switch in efficacy of DMCM, the convulsant action of DMCM was examined in wild type and 1(H101R) mice.
In contrast DMCM failed to elicit convulsions in 1(H101R) mice convulsed (p 0.001 compared to wild type mice) (Fig.
Discussion The present results can be summarized as follows: (1) In wild type mice, the benzodiazepine inverse agonist Ro 15-4513 at the dose of 10 mg/kg stimulated baseline locomotion and displayed a proconvulsant action when combined with pentylenetetrazole; (2) In contrast, in 1(H101R) mice the same treatment with Ro 15-4513 decreased locomotor activity and did not potentiate pentylenetetrazole convulsions; (3) At a higher dose (30 mg/kg) Ro 15-4513 acted as an anticonvulsant agent in 1(H101R) mice.
Furthermore the sensitivity to pentylenetetrazole convulsions was unaltered in the mutant mice; (4) The -carboline DMCM (1 M) acted as an inverse agonist at recombinant 122 receptors whereas it displayed an agonistic effect in potentiating relative GABA-evoked chloride currents at mutated 1(H101R)22 receptors; and, (5) DMCM at the dose of 6 mg/kg elicited convulsions in wild type mice whereas it was inactive in 1(H101R) mice.
As the 1-GABAA receptor is the most abundant GABAA receptor subtype in adult brain (Fritschy and Mohler, 1995), the 1(H101R) mice with a high prevalence of mutated 1-receptors provided a suitable animal model to examine in vivo the consequences of the H101R point mutation-induced switch in pharmacological efficacy of Ro 15-4513.
We have previously reported on the basis of the analysis of the 1(H101R) mice that the motor inhibitory and anticonvulsant actions of two benzodiazepine site agonists, diazepam and zolpidem are mediated by the 1-GABAA receptors (Rudolph et al., 1999; Crestani et al., 2000; McKernan et al., 2000).
Ro 15-4513 not only failed to produce these two inverse agonistic effects but rather displayed agonistic-like motor depressant and anticonvulsant properties in 1(H101R) mice.
Moreover they confirm in vivo previous observations on recombinant diazepam-insensitive 1(H101R)-GABAA receptors (Kleingoor et al., 1993; Benson et al., 1998); that is a H101R point mutation in the 1-subunit of GABAA receptors switches the pharmacological efficacy of Ro 15-4513 from inverse agonism to agonism.
Indeed, we have shown in vitro that the inverse agonistic effect of DMCM at recombinant wild type GABAA receptors switched to an agonistic effect at 1(H101R)GABAA receptors.
In vivo, DMCM failed to elicit convulsions in 1(H101R) mice.
The lack of response to convulsant drugs in 1(H101R) mice was restricted to benzodiazepine site ligands since the susceptibility to pentylenetetrazole-induced convulsions was unaltered in these mutants.
Convulsant action of DMCM in wild type and 1(H101R) mice.
11278406
full text
L272F
protein
substitution
true negative
(19) is L272F.) The mutation is located in transmembrane segment S5, indicating a possible involvement of the S5 region in channel inactivation.
L273F
protein
substitution
P51787
true positive
The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation.
Here we show that w h e n e x p r e s s i n g m u t a n t I Ks c h a n n e l s f o r m e d f r o m KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation.
The LQT1 mutation L273F (8) exhibited a pronounced macroscopic inactivation when expressed in Xenopus oocytes (19).
Mutation G272C, adjacent to the site of clinical mutation L273F (as described above), caused additionally slightly slowed activation kinetics.
Introducing a phenylalanine at position 243 in KCNQ2 resulted in inactivating currents interestingly similar to the KCNQ1 mutant L273F (Fig.
Asterisk indicates the site of the LQT1 mutant L273F.
We next examined the pathophysiological role of KCNQ1 inactivation by studying kinetics of the LQT1 mutant L273F, which is located next to Gly272 and in the KcsA channel model in close proximity to Val307.
The mutant was analyzed previously (19); the authors reported a pronounced macroscopic in- activation of KCNQ1(L273F) as well as a reduced current amplitude of KCNQ1(L273F)/MinK compared with KCNQ1-WT/ MinK.
The greatly enhanced inactivation of KCNQ1(L273F) is shown in Fig.
After coinjecting the same amounts of KCNQ1-WT or KCNQ1(L273F) cRNA together with MinK cRNA, we observed a decreased current amplitude for mutant IKs channels consistent with previous data (19) (Fig.
Most interestingly, using the double-pulse protocol, we demonstrated that MinK is no longer able to completely abolish the pronounced inactivation in KCNQ1(L273F) (Fig.
Moreover, this inactivation in mutant IKs channels was accelerated compared with homomeric KCNQ1(L273F), further indicating functional assembly of the mutant KCNQ1 subunit with MinK (Table II).
Tail currents of KCNQ1(L273F)/MinK channels displayed a weak hook confirming the persistence of inactivation in these heteromers (Fig.
nonconducting, mutant IKs channels contributes to the decreased amplitudes of KCNQ1(L273F) and KCNQ1(L273F)/MinK com- mV increments in an oocyte injected with cRNA of the respective construct shown in B.
Characterization of the LQT1 mutant L273F in oocytes.
B, current traces of a L273F-coinjected oocyte recorded as described in A.
D, current traces of a L273F/MinK-coinjected oocyte recorded as described in A.
E, IV relationship of mutant L273F/MinK (n 14) and of KCNQ1-WT/MinK (n 17) from oocytes recorded as in B and D.
F, The fraction of inactivated channels of mutant L273F/MinK and of KCNQ1WT/MinK was determined by tail current analysis (double exponential fit) and calculated by the amplitudes of recovery from inactivation (Afast) and deactivation (Aslow) (see "Experimental Procedures").
In conclusion, both C-type and KCNQ1 inactivation occur within the pore Geography of KCNQ1 Inactivation TABLE II Characteristics of inactivation in KCNQ1-WT and KCNQ1(L273F) in homomeric and heteromeric expression together with MinK in Xenopus oocytes Oocytes were injected with 10 ng of KCNQ1 cRNA.
T365A
protein
substitution
true negative
Point mutants V308I and V310L and a double mutant T365A/L366W were still inactivating (data not shown).
V310L
protein
substitution
P51787
true positive
Point mutants V308I and V310L and a double mutant T365A/L366W were still inactivating (data not shown).
G272T
protein
substitution
true positive
P51787
Now, activation and deactivation kinetics of G272T were very similar to KCNQ1-WT (Table I); however inactivation still was completely abolished.
Constants WT-KCNQ1 KCNQ1(G272C) KCNQ1(G272T) KCNQ1(V307L) Activation, fast Activation, slow Deactivation, fast Deactivation, slow 33 0.85 222 1.7 3 ms (9) 0.09 s (9) 2 ms (9) 0.4 s (9) 103 0.68 267 7 ms (9) 0.05 s (9) 18 ms (9) -- 41 0.27 238 6.5 2 ms (8) 0.0 s (8) 4 ms (8) 0.1 s (8) 76 0.67 420 4 ms (9) 0.06 s (9) 30 ms (9) -- FIG.
L243F
protein
substitution
true negative
We constructed the KCNQ2 mutations C242G, L243F, and L272V.
In a construct containing both mutations L243F and L272V, an inactivation was apparent although the currents were small in amplitude (Fig.
A, B, and C, models of the KCNQ2 channel subunit including introduced mutations L243F (A), L272V (B), and L243F/L272V are drawn on the left side (C).
V307L
protein
substitution
P51787
true positive
Expression of these point mutants showed that two single amino acid substitutions, G272C and V307L, are capable of abolishing inactivation (Fig.
The other key mutation V307L located in the pore loop showed slightly slowed activation and deactivation kinetics compared with KCNQ1-WT (Table I), but again abolished inactivation.
Constants WT-KCNQ1 KCNQ1(G272C) KCNQ1(G272T) KCNQ1(V307L) Activation, fast Activation, slow Deactivation, fast Deactivation, slow 33 0.85 222 1.7 3 ms (9) 0.09 s (9) 2 ms (9) 0.4 s (9) 103 0.68 267 7 ms (9) 0.05 s (9) 18 ms (9) -- 41 0.27 238 6.5 2 ms (8) 0.0 s (8) 4 ms (8) 0.1 s (8) 76 0.67 420 4 ms (9) 0.06 s (9) 30 ms (9) -- FIG.
A and B, model of the chimera Q2S5-S6Q1 including introduced mutations G272C (A) and V307L (B).
L272V
protein
substitution
true negative
We constructed the KCNQ2 mutations C242G, L243F, and L272V.
The mutant KCNQ2(L272V) did not exert a KCNQ1like inactivation as shown in Fig.
In a construct containing both mutations L243F and L272V, an inactivation was apparent although the currents were small in amplitude (Fig.
A, B, and C, models of the KCNQ2 channel subunit including introduced mutations L243F (A), L272V (B), and L243F/L272V are drawn on the left side (C).
C242G
protein
substitution
true negative
We constructed the KCNQ2 mutations C242G, L243F, and L272V.
G272C
protein
substitution
true positive
P51787
Expression of these point mutants showed that two single amino acid substitutions, G272C and V307L, are capable of abolishing inactivation (Fig.
Mutation G272C, adjacent to the site of clinical mutation L273F (as described above), caused additionally slightly slowed activation kinetics.
Constants WT-KCNQ1 KCNQ1(G272C) KCNQ1(G272T) KCNQ1(V307L) Activation, fast Activation, slow Deactivation, fast Deactivation, slow 33 0.85 222 1.7 3 ms (9) 0.09 s (9) 2 ms (9) 0.4 s (9) 103 0.68 267 7 ms (9) 0.05 s (9) 18 ms (9) -- 41 0.27 238 6.5 2 ms (8) 0.0 s (8) 4 ms (8) 0.1 s (8) 76 0.67 420 4 ms (9) 0.06 s (9) 30 ms (9) -- FIG.
A and B, model of the chimera Q2S5-S6Q1 including introduced mutations G272C (A) and V307L (B).
V308I
protein
substitution
P51787
true positive
Point mutants V308I and V310L and a double mutant T365A/L366W were still inactivating (data not shown).
L366W
protein
substitution
true negative
Point mutants V308I and V310L and a double mutant T365A/L366W were still inactivating (data not shown).
11959905
full text
L92M
protein
substitution
true negative
Four additional c onstr ucts were made to assess the role of the leucine residue af ter the predicted filter glycine in P-loop A: AtHKT1-M69L, AtHKT1-S68G-M69L, HKT1-L92M, and HKT1-G91S-L92M.
Only AtHKT1-S68G-M69L and HKT1-L92M allowed g row th at 1 mM K , the c onstr ucts w ith a glycine at the predicted filter position.
HKT1-L92M rendered G19 cells as Na sensitive as HKT1, and HKT1-G91S-L92M rendered them as Na sensitive as HKT1-G91S.
HKT1-G91S and HKT1-G91S-L92M did not elicit any Na or K currents, indicating that these c onstr ucts may not be properly ex pressed in Xenopus ooc y tes (dat a not shown).
The reverse mut ation in HKT1-L92M abolished this dampen ing ef fect of K on Maser et al.
Only transporters with a glycine at the filter position in P-loop A (HKT1, AtHKT1-S68G, HKT1-L92M) were more permeable to K as indicated by large positive shifts in the reversal potential with increasing external [K ].
G91S
protein
substitution
true negative
However, AtHKT1-S68G was not as ef fective in suppressing the trk1,2 phenot ype as was HKT1, and HKT1-G91S still promoted g row th of CY162 cells better than did AtHKT1 (Fig.
Four additional c onstr ucts were made to assess the role of the leucine residue af ter the predicted filter glycine in P-loop A: AtHKT1-M69L, AtHKT1-S68G-M69L, HKT1-L92M, and HKT1-G91S-L92M.
Mut ation of Gly-91 to serine (HKT1-G91S) markedly increased the salt sensitiv it y to about the same extent as AtHKT1.
HKT1-L92M rendered G19 cells as Na sensitive as HKT1, and HKT1-G91S-L92M rendered them as Na sensitive as HKT1-G91S.
The c onverse mut ation in HKT1, Gly-91 to serine (HKT1-G91S), reduced c omplement ation of the trk1,2 phenot ype (Fig.
HKT1-G91S and HKT1-G91S-L92M did not elicit any Na or K currents, indicating that these c onstr ucts may not be properly ex pressed in Xenopus ooc y tes (dat a not shown).
G88S
protein
substitution
true negative
Mutation of Ser-88 to glycine in OsHKT1 (OsHKT1-S88G) restores K -uptake complementation, whereas mutation of Gly-88 to serine in OsHKT2 (OsHKT2-G88S) abrogates K -uptake complementation.
To test further the hypothesis that the presence of glycine in P-loop A deter mines K per meabilit y, we mut ated Ser-88 of OsHKT1 to glycine (OsHKT1-S88G) and Gly-88 of OsHKT2 to serine (OsHKT2-G88S).
OsHKT2-G88S, in c ontrast to OsHKT2, did not per mit g row th at low [K ] (Fig.
S88G
protein
substitution
true negative
Mutation of Ser-88 to glycine in OsHKT1 (OsHKT1-S88G) restores K -uptake complementation, whereas mutation of Gly-88 to serine in OsHKT2 (OsHKT2-G88S) abrogates K -uptake complementation.
To test further the hypothesis that the presence of glycine in P-loop A deter mines K per meabilit y, we mut ated Ser-88 of OsHKT1 to glycine (OsHKT1-S88G) and Gly-88 of OsHKT2 to serine (OsHKT2-G88S).
CY162 cells ex pressing OsHKT1 did not grow on 100 M K , whereas OsHKT1-S88G ex pressing cells were able to g row on 100 M K (Fig.
M69L
protein
substitution
true negative
Four additional c onstr ucts were made to assess the role of the leucine residue af ter the predicted filter glycine in P-loop A: AtHKT1-M69L, AtHKT1-S68G-M69L, HKT1-L92M, and HKT1-G91S-L92M.
Only AtHKT1-S68G-M69L and HKT1-L92M allowed g row th at 1 mM K , the c onstr ucts w ith a glycine at the predicted filter position.
AtHKT1-S68G and AtHKT1-S68G-M69L did not support g row th of CY162 cells at low [K ] in the absence of Na (Fig.
Calculation of K to Na per meabilit y ratios (PK PNa ) (15) showed values of 40.8 for HKT1, 0.07 for AtHKT1, 4.7 for AtHKT1-S68G, and 0.033 for AtHKT1-M69L.
The mut ation AtHKT1-M69L did not cause pot assium-dependent shif ts in the reversal potential, c onsistent w ith lack of c omplement ation of K deficienc y by AtHKT1-M69L (Figs.
However, AtHKT1-M69L-mediated out ward currents were sensitive to K .
AtHKT1-M69L-mediated out ward currents decreased w ith increasing [K ] (Fig.
Y162S
protein
substitution
true negative
For c omplement ation of the CY162 S.
S68G
protein
substitution
true negative
Thus, substitution of a single amino acid, Ser-68 to Gly, is suf ficient to restore K transport to AtHKT1 as measured by rescue of CY162 cells.
10619558
full text
10962018
full text
A700X
protein
substitution
true positive
P35523
Cells expressing WT as well as G750X, A700X, and L590X hClC-1 channels showed a staining of the surface membrane (Fig.
5A shows mean current amplitudes for five distinct carboxyl-terminal truncations that are non-functional when expressed alone, L590X, G650X, A700X, G750X, and E800X.
A700X ClC-1 and mutant ClC-1 with larger carboxyl termini were rescued by co-expressing various carboxyl-terminal proteins (Fig.
Co-transfection of A700X, G750X, and E800X with Cterm(E800-L988) did not result in the appearance of a ClC-1 current component, which demonstrates the importance of the linker between amino acids 700 and 800 for this interaction.
Moreover, in co-expression experiments CBS1 and CBS2 were not equivalent because co-expressing A700X hClC-1 with Cterm(A700-L988CBS1) did not result in the formation of functional channels (Fig.
BG, confocal images of living cells transfected with Cterm(L590L988)-CFP (B), Cterm(A700-L988)-CFP (C), Cterm(A700-L988CBS1)-CFP (D), YFP-L590X hClC-1 and Cterm(L590-L988)-CFP (E), YFP-A700X hClC-1 and Cterm(A700-L988)-CFP (F), and YFP- (D607-Q662) hClC-1 and Cterm(A700-L988CBS1)-CFP (G) were taken 48 h after transfection.
For example, in cells cotransfected with YFP-A700X hClC-1 and Cterm(A700-L988)CFP the majority of CFP fluorescence is overlaid to the YFP fluorescence (visible as orange color in Fig.
5F), indicating a stable binding of YFP-A700X hClC-1 and Cterm(A700-L988)CFP.
In contrast, the carboxylterminal fusion protein Cterm(L590-L988)-CFP is redistributed by YFP-A700X hClC-1 (Fig.
A and B, confocal images of living cells transfected with YFP-hClC-1 and Cterm(A700-L988)CFP (A), YFP-hClC-1 and Cterm(A700L988CBS1)-CFP (B), and YFP-A700X hClC-1 and Cterm(L590-L988)-CFP (C) were taken 48 h after transfection.
Moreover, no colocalization of A700X and G750X hClC-1 (containing CBS1) with Cterm(E800-L988) (containing CBS2) was observed (Table I).
The isolated carboxyl terminus (Cterm(L590-L988)) is capable of binding to A700X hClC-1 and of relocalization to the membrane (Fig.
G750X
protein
substitution
true positive
P35523
Cells expressing WT as well as G750X, A700X, and L590X hClC-1 channels showed a staining of the surface membrane (Fig.
5A shows mean current amplitudes for five distinct carboxyl-terminal truncations that are non-functional when expressed alone, L590X, G650X, A700X, G750X, and E800X.
Co-transfection of A700X, G750X, and E800X with Cterm(E800-L988) did not result in the appearance of a ClC-1 current component, which demonstrates the importance of the linker between amino acids 700 and 800 for this interaction.
Comparing expression levels for various transmembrane constructs with a given carboxyl terminus, G750X exhibited the highest expression levels of all combinations (Fig.
When transfected with Cterm(G750-L988), no significant difference in the current amplitudes for G750X and E800X were observed (Fig.
Moreover, no colocalization of A700X and G750X hClC-1 (containing CBS1) with Cterm(E800-L988) (containing CBS2) was observed (Table I).
Y850X
protein
substitution
true positive
P35523
In cells expressing Y850X hClC-1 channels, the staining of the surface membrane was decreased, suggesting that this mutation might also interfere with surface membrane insertion.
S886X
protein
substitution
true positive
P35523
Image mutation
K875X
protein
substitution
true positive
P35523
Image mutation
G700X
protein
substitution
true negative
Although separate segments of the WT carboxyl terminus bind to each other, preventing an interaction of the soluble fusion protein with WT-L590X hClC-1 heterodimers, Cterm(A700-L988)-CFP can 13146 The Carboxyl Terminus of hClC-1 TABLE I Co-localization of C-terminal and transmembrane fluorescent fusion proteins Soluble CFP constructs Transmembrane YFP constructs Cterm(L590-L988) Cterm(A700-L988) Cterm(A700-L988CBS1) Cterm(E800-L988) A590X hC1C-1 L G700X hC1C-1 E 750X hC1C-1 Y800X hC1C-1 850X hC1C-1 (D607-Q662) hC1C-1 W (D607-Q662)-E800X hC1C-1 a T hC1C-1 b c a b NDc ND ND ND ND ND , no co-localization.
E800X
protein
substitution
true positive
P35523
5A shows mean current amplitudes for five distinct carboxyl-terminal truncations that are non-functional when expressed alone, L590X, G650X, A700X, G750X, and E800X.
Co-transfection of A700X, G750X, and E800X with Cterm(E800-L988) did not result in the appearance of a ClC-1 current component, which demonstrates the importance of the linker between amino acids 700 and 800 for this interaction.
When transfected with Cterm(G750-L988), no significant difference in the current amplitudes for G750X and E800X were observed (Fig.
We designed a concatameric construct in which one hClC-1 subunit lacking CBS1 (YFP- (D607-Q662) hClC-1) was linked to E800X hClC-1 (Fig.
Although separate segments of the WT carboxyl terminus bind to each other, preventing an interaction of the soluble fusion protein with WT-L590X hClC-1 heterodimers, Cterm(A700-L988)-CFP can 13146 The Carboxyl Terminus of hClC-1 TABLE I Co-localization of C-terminal and transmembrane fluorescent fusion proteins Soluble CFP constructs Transmembrane YFP constructs Cterm(L590-L988) Cterm(A700-L988) Cterm(A700-L988CBS1) Cterm(E800-L988) A590X hC1C-1 L G700X hC1C-1 E 750X hC1C-1 Y800X hC1C-1 850X hC1C-1 (D607-Q662) hC1C-1 W (D607-Q662)-E800X hC1C-1 a T hC1C-1 b c a b NDc ND ND ND ND ND , no co-localization.
A and B, confocal image of a living cell expressing YFP(D607-Q662)-E800X hClC-1 heterodimers and Cterm(A700-L988)-CFP (A) and of YFP-WT-L590X hClC-1 heterodimers and Cterm(A700-L988)-CFP (B).
bind to YFP- (D607-Q662)-E800X hClC-1 heterodimers, indicating an accessible binding site within the carboxyl termini.
G650X
protein
substitution
true positive
P35523
5A shows mean current amplitudes for five distinct carboxyl-terminal truncations that are non-functional when expressed alone, L590X, G650X, A700X, G750X, and E800X.
S537F
protein
substitution
true positive
P35523
We engineered truncated ClC-1 subunits carrying a point mutation (S537F) rendering mutant hClC-1 channels insensitive to block by 9-anthracene carboxylic acid (9-AC) (29, 30).
If both protopores were functional in heterodimeric channels with only one carboxyl terminus, one would expect a similar relative current reduction by 9-AC in cells expressing WT-S537F and in cells expressing heterodimeric channels in which one of the two carboxyl termini is deleted, i.e.
WT-S537F/L590X and S537FL590X hClC-1 channels.
4A shows current recordings of a tsA201 cell expressing a WT-S537F/ 13144 The Carboxyl Terminus of hClC-1 FIG.
A, inhibitory effects of 0.2 mM 9-anthracene carboxylic acid on WT-S537F/L590X hClC-1 heterodimeric channels.
C, relative current block of WT hClC-1, S537F hClC-1, WT-S537F/L590X hClC-1, S537F-L590X hClC-1, and WT-S537F hClC-1 channels by 0.2 mM 9-anthracene carboxylic acid.
The relative block of WT-S537F/ L590X hClC-1 channels (blocked current fraction: 0.80 0.01, n 15, p 0.01) was significantly larger, and the relative block observed for S537F-L590X heterodimers (0.29 0.02, n 12, p 0.01) significantly smaller than the relative block of WT-S537F hClC-1 (0.55 0.03, n 8) (Fig.
This demonstrates that the protopore without attached carboxyl terminus is non-functional in the WT-S537F/L590X as well as in the S537F-L590X hClC-1 heterodimer.
L590X
protein
substitution
true positive
P35523
Cells expressing WT as well as G750X, A700X, and L590X hClC-1 channels showed a staining of the surface membrane (Fig.
Representative whole-cell current traces recorded from tsA201 cells expressing WT (A), (D607-Q662)-L590X heterodimeric (B), S132C homodimeric (D), WT-S132C heterodimeric (E), and S132C-L590X heterodimeric hClC-1 channels (F).
Heterodimeric WT-L590X hClC-1 channels containing only one complete carboxyl terminus and even (D607-Q662)-L590X hClC-1 channels with only one CBS domain both form functional channels with gating and permeation properties similar to those of WT (Fig.
Expression of a concatameric construct linking S132C hClC-1 with L590X hClC-1 in tsA201 cells gives rise to the expression of an anion current whose gating properties are similar to those of heterodimeric WT-S132C hClC-1 channels (Fig.
Because L590X hClC-1 homodimeric channels are non-functional, currents that deactivate upon hyperpolarizing voltage steps must be conducted by heterodimeric channels with only one carboxyl terminus (Fig.
WT-S537F/L590X and S537FL590X hClC-1 channels.
A, inhibitory effects of 0.2 mM 9-anthracene carboxylic acid on WT-S537F/L590X hClC-1 heterodimeric channels.
C, relative current block of WT hClC-1, S537F hClC-1, WT-S537F/L590X hClC-1, S537F-L590X hClC-1, and WT-S537F hClC-1 channels by 0.2 mM 9-anthracene carboxylic acid.
L590X hClC-1 heterodimeric construct after application of 0.2 mM 9-AC in the external solution.
The relative block of WT-S537F/ L590X hClC-1 channels (blocked current fraction: 0.80 0.01, n 15, p 0.01) was significantly larger, and the relative block observed for S537F-L590X heterodimers (0.29 0.02, n 12, p 0.01) significantly smaller than the relative block of WT-S537F hClC-1 (0.55 0.03, n 8) (Fig.
This demonstrates that the protopore without attached carboxyl terminus is non-functional in the WT-S537F/L590X as well as in the S537F-L590X hClC-1 heterodimer.
5A shows mean current amplitudes for five distinct carboxyl-terminal truncations that are non-functional when expressed alone, L590X, G650X, A700X, G750X, and E800X.
BG, confocal images of living cells transfected with Cterm(L590L988)-CFP (B), Cterm(A700-L988)-CFP (C), Cterm(A700-L988CBS1)-CFP (D), YFP-L590X hClC-1 and Cterm(L590-L988)-CFP (E), YFP-A700X hClC-1 and Cterm(A700-L988)-CFP (F), and YFP- (D607-Q662) hClC-1 and Cterm(A700-L988CBS1)-CFP (G) were taken 48 h after transfection.
No redistribution and no overlay could be observed for the two complementary fragments YFP-L590X hClC-1 and Cterm(L590-L988)-CFP (Fig.
In contrast, a heterodimer consisting of one YFP-WT hClC-1 and one L590X hClC-1 subunit does not bind Cterm(A700-L988)-CFP (Fig.
Although separate segments of the WT carboxyl terminus bind to each other, preventing an interaction of the soluble fusion protein with WT-L590X hClC-1 heterodimers, Cterm(A700-L988)-CFP can 13146 The Carboxyl Terminus of hClC-1 TABLE I Co-localization of C-terminal and transmembrane fluorescent fusion proteins Soluble CFP constructs Transmembrane YFP constructs Cterm(L590-L988) Cterm(A700-L988) Cterm(A700-L988CBS1) Cterm(E800-L988) A590X hC1C-1 L G700X hC1C-1 E 750X hC1C-1 Y800X hC1C-1 850X hC1C-1 (D607-Q662) hC1C-1 W (D607-Q662)-E800X hC1C-1 a T hC1C-1 b c a b NDc ND ND ND ND ND , no co-localization.
A and B, confocal image of a living cell expressing YFP(D607-Q662)-E800X hClC-1 heterodimers and Cterm(A700-L988)-CFP (A) and of YFP-WT-L590X hClC-1 heterodimers and Cterm(A700-L988)-CFP (B).
However, an isolated complete hClC-1 carboxyl terminus (Cterm(L590L988)) does not co-localize with a L590X hClC-1, indicating that the carboxyl terminus does not bind directly to a transmembrane domain (Fig.
E865X
protein
substitution
true positive
P35523
In contrast, deletions of parts of CBS2, even as little as 6 amino acids (E865X), caused a complete disappearance of the ClC-1 current component (Fig.
R894X
protein
substitution
true positive
P35523
Image mutation
Y800X
protein
substitution
true negative
Although separate segments of the WT carboxyl terminus bind to each other, preventing an interaction of the soluble fusion protein with WT-L590X hClC-1 heterodimers, Cterm(A700-L988)-CFP can 13146 The Carboxyl Terminus of hClC-1 TABLE I Co-localization of C-terminal and transmembrane fluorescent fusion proteins Soluble CFP constructs Transmembrane YFP constructs Cterm(L590-L988) Cterm(A700-L988) Cterm(A700-L988CBS1) Cterm(E800-L988) A590X hC1C-1 L G700X hC1C-1 E 750X hC1C-1 Y800X hC1C-1 850X hC1C-1 (D607-Q662) hC1C-1 W (D607-Q662)-E800X hC1C-1 a T hC1C-1 b c a b NDc ND ND ND ND ND , no co-localization.
pdftotext error
A590X
protein
substitution
true negative
Although separate segments of the WT carboxyl terminus bind to each other, preventing an interaction of the soluble fusion protein with WT-L590X hClC-1 heterodimers, Cterm(A700-L988)-CFP can 13146 The Carboxyl Terminus of hClC-1 TABLE I Co-localization of C-terminal and transmembrane fluorescent fusion proteins Soluble CFP constructs Transmembrane YFP constructs Cterm(L590-L988) Cterm(A700-L988) Cterm(A700-L988CBS1) Cterm(E800-L988) A590X hC1C-1 L G700X hC1C-1 E 750X hC1C-1 Y800X hC1C-1 850X hC1C-1 (D607-Q662) hC1C-1 W (D607-Q662)-E800X hC1C-1 a T hC1C-1 b c a b NDc ND ND ND ND ND , no co-localization.
pdftotext error
S132C
protein
substitution
true positive
P35523
Representative whole-cell current traces recorded from tsA201 cells expressing WT (A), (D607-Q662)-L590X heterodimeric (B), S132C homodimeric (D), WT-S132C heterodimeric (E), and S132C-L590X heterodimeric hClC-1 channels (F).
To test whether the expression of heterodimeric concatamers supports the formation of homodimers by such a mechanism, we inserted a naturally occurring, disease-causing point mutation, S132C (28), in one of the two subunits.
S132C modifies gating properties of ClC-1 in homodimeric S132C hClC-1 and in heterodimeric WT- S132C hClC-1 channels in a different way (Fig.
Homodimeric S132C hClC-1 channels activate upon membrane hyperpolarization (Fig.
3D), whereas expression of WT-S132C hClC-1 concatamers results in a depolarization-activated anion cur- rent that differs from homodimeric WT channels in voltage dependence and in minimum open probability at negative potentials (Fig.
The absence of a hyperpolarizationactivated current amplitude indicates that homodimeric S132C hClC-1 channels are not formed under these conditions and demonstrates that expression of concatamers results in a homogenous population of heterodimeric anion channels.
Expression of a concatameric construct linking S132C hClC-1 with L590X hClC-1 in tsA201 cells gives rise to the expression of an anion current whose gating properties are similar to those of heterodimeric WT-S132C hClC-1 channels (Fig.
15326291
full text
Y143H
protein
substitution
true negative
This finding is import ant because the -chair lacks the S278 and Y143 H-bonds in the A pocket for med by the -chair (Fig.
Y143F
protein
substitution
true negative
CF RCI and genomic transactivation (EC50 values) in VDRwt and targeted VDR hydroxyl mutants Genomic transactivation, EC50 VDR construct RCI for CF, % 13 20 41 (n 1.6 1.9 2) 94 1,25D, nM 1.0 (n 0.3 (n 34 (n 23) 5.4 5) 0.88 3) 1.3 CF, nM 1.3 (n 3) 0.29 (n 3) 0.3 (n 3) VDRwt S278A Y143F S278A 130 Fig.
We made S278A and Y143F S278A mut ant VDRs to test the ensemble model w ith 1,25D and CF (Fig.
The Y143F S278A mut ation had the same ef fect on CF's EC50 obser ved in S278A; however, there was an 10-fold increase in CF's RCI (Table 2).
This result can be ex plained by the Y143F S278A mut ation af fecting 1,25D's af fin it y for the G pocket more than its st abilit y in the A pocket (loss of 7 kcal mol and 3 kcal mol in the calculated IE, respectively; Table 3).
Import antly, if no A pocket ex isted, then, based on CF's Y143F S278A RCI being 100, CF and 1,25D should transactivate w ith nearly the same ef ficac y in the double mut ant; however, this result is not supported by the dat a (Table 2).
S278A
protein
substitution
true negative
The VDR mut ant c onstr uct S278A was a gif t f rom E.
CF RCI and genomic transactivation (EC50 values) in VDRwt and targeted VDR hydroxyl mutants Genomic transactivation, EC50 VDR construct RCI for CF, % 13 20 41 (n 1.6 1.9 2) 94 1,25D, nM 1.0 (n 0.3 (n 34 (n 23) 5.4 5) 0.88 3) 1.3 CF, nM 1.3 (n 3) 0.29 (n 3) 0.3 (n 3) VDRwt S278A Y143F S278A 130 Fig.
We made S278A and Y143F S278A mut ant VDRs to test the ensemble model w ith 1,25D and CF (Fig.
The S278A mut ation has no sign ificant ef fect on CF's RCI, but causes a 5 - f o l d d e c r e a s e i n C F ' s g e n o m i c E C 50 ( T a b l e 2 ) ; h o w e v e r , S 2 7 8 A did not af fect 1,25D's EC50 (Table 2).
The Y143F S278A mut ation had the same ef fect on CF's EC50 obser ved in S278A; however, there was an 10-fold increase in CF's RCI (Table 2).
This result can be ex plained by the Y143F S278A mut ation af fecting 1,25D's af fin it y for the G pocket more than its st abilit y in the A pocket (loss of 7 kcal mol and 3 kcal mol in the calculated IE, respectively; Table 3).
Thus, the 10-fold increase in CF's RCI c ompared w ith S278A and VDRw t can be ex plained by CF's c omplete loss of A pocket st abilit y, which strengthens its G pocket selectiv it y ( IE 21 kcal mol), whereas 1,25D's G pocket selectiv it y is reduced by the double mut ation ( IE 13 kcal mol), supporting its low EC50 (Table 2).
Import antly, if no A pocket ex isted, then, based on CF's Y143F S278A RCI being 100, CF and 1,25D should transactivate w ith nearly the same ef ficac y in the double mut ant; however, this result is not supported by the dat a (Table 2).
10962010
full text
S435F
protein
substitution
true positive
Q9NQW8
The purpose was to examine the functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G).
Macroscopic patch currents were recorded from human embryonic kidney (HEK) 293 cells expressing homomeric (hCNGA3 and hCNGB3) and heteromeric (hCNGA3/ hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3D633G) channels using inside-out patch-clamp technique.
The selectivity of hCNGA3, hCNGA3/ hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3D633G channels for monovalent cations were largely similar.
The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at From the Departments of 1Ophthalmology, 2Medical Biochemistry, and 3Physiology, Shiga University of Medical Science, Seta, Otsu, Japan; and 4Institute of Vision Research, Nagoya, Japan.
Furthermore, we characterized the functional consequences of rod monochromacy-associated S435F6,12 and D633G mutations in hCNGB3 in a similar way.
Two mutations, 1304C T (S435F) and 1868A G (D633G), designated hCNGB3-S435F and hCNGB3-D633G, respectively, were each introduced to the hCNGB3 subunit cDNA as previously described,16 and these mutant cDNAs were also cloned into pCR3.1.
The amounts of each vector were ( g/ dish): 1.5 hCNGA3 and 0.5 GFP or 0.75 hCNGA3, 0.75 hCNGB3 (or hCNGB3-S435F, hCNGB3-D633G) and 0.5 GFP.
The activation of hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3S435F, and hCNGA3/hCNGB3-D633G channels typically exhibited little if any rundown or runup phenomena during exposure to various concentrations (2100 M) of cGMP for a period of at least 20 minutes.
The hCNGB3, hCNGB3S435F, and hCNGB3-D633G subunit cDNAs were cloned into the KpnI site of pFLAG-CMV plasmid (Sigma Chemical Co., St.
7 Rod Monochromacy-Associated hCNGB3 Mutations 2327 Effects of Rod Monochromacy-Associated Mutations in hCNGB3 on Apparent Affinity for cGMP in Heteromeric Channels The cGMP sensitivity of the heteromeric channels composed of hCNGA3 and rod monochromacy-associated mutants of hCNGB3 (hCNGB3-D633G and hCNGB3-S435F) was characterized to clarify the functional consequences of these two mutations in the hCNGB3.
4A) and hCNGA3/hCNGB3-S435F (Fig.
Figures 4C and 4D, respectively, represent the concentrationresponse relationships for the activation of hCNGA3/hCNGB3-D633G and hCNGA3/hCNGB3S435F heteromeric channels by cGMP.
Figure 5 illustrates representative IV relationships for hCNGA3 (A), hCNGA3/ hCNGB3 (B), hCNGA3/hCNGB3-D633G (C), and hCNGA3/ hCNGB3-S435F (D) channels activated by 100 M cGMP under the various bi-ionic solutions.
Macropatch currents activated by various concentrations of cGMP, recorded from HEK293 cells expressing hCNGA3/hCNGB3D633G (A) and hCNGA3/hCNGB3-S435F (B) heteromeric channels.
Concentrationresponse relationships for the activation of hCNGA3/ hCNGB3-D633G (C, filled squares) and hCNGA3/hCNGB3-S435F (D, open squares) heteromeric channels.
12.0 2.1 M and nH of 1.85 0.12 for hCNGA3/hCNGB3S435F (n 10; Fig.
The K1/2 value for the activation of both hCNGA3/hCNGB3-D633G and hCNGA3/hCNGB3-S435F is thus similar to that for hCNGA3 homomers (11.1 1.0 M) but is significantly smaller than that for hCNGA3/hCNGB3 heteromers (26.2 1.9 M, P 0.001).
Immunoprecipitation experiments showed that both mutants of hCNGB3 (hCNGB3-D633G and hCNGB3-S435F) were associated with the hCNGA3 subunit (Fig.
Panels A, B, C, and D represent the current traces recorded from hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-D633G, and hCNGA3/hCNGB3-S435F channels, respectively.
Relative Permeability and Conductance Ratios of Wild-Type and Mutant Channels for Monovalent Cations Relative Permeability Ratio (PC/PK) h Relative Conductance Ratio (GC/GK) C K 1.0 1.0 1.0 1.0 N a 0.01 0.01 0.01 0.02 L i 0.02 0.03 0.08 0.04 R b 0.02 0.02 0.04 0.03 s 0.03 0.05 0.11 0.03 K Na 1.70 1.12 1.43 1.84 0.06 0.06 0.09 0.04 L i 0.01 0.01 0.01 0.08 R b 0.02 0.02 0.04 0.04 C s 0.01 0.03 0.06 0.06 CNGA3 hCNGA3/hCNGB3 hCNGA3/hCNGB3-D633G hCNGA3/hCNGB3-S435F Data are mean values 0.96 1.00 0.97 0.98 0.76 0.88 0.75 0.77 0.84 0.83 0.87 0.77 0.51 0.61 0.51 0.46 1.0 1.0 1.0 1.0 0.57 0.57 0.56 0.60 0.38 0.58 0.51 0.36 0.27 0.40 0.28 0.23 SEM from 4 8 patches.
(hCNGA3) and heteromeric (hCNGA3/hCNGB3, hCNGA3/ hCNGB3-D633G and hCNGA3/hCNGB3-S435F) channels were not appreciably affected (data not shown).
The present study further characterized the functional consequences of the rod monochromacy-associated mutations in hCNGB3 (S435F and D633G).
However, the heteromeric channels composed of hCNGA3 and rod monochromacy-associated mutants of hCNGB3 (hCNGB3-D633G and hCNGB3-S435F) exhibited a higher apparent affinity for cGMP compared with wild-type heteromeric channels (Fig.
This functional alteration in cone CNG channels associated with D633G or S435F mutation in hCNGB3 should cause the channels to be reluctant to the decrease in intracellular cGMP level after reception of photons, leading to their being always open, which should be responsible at least partly for the pathogenesis of rod monochromacy.
recently reported a similar increase in the apparent affinity for cGMP in the rod monochromacy-associated S435F mutation in hCNGB3 subunits.26 Serine at position 435 resides in the transmembrane segment S6 (indicated by 1, Fig.
The sensitivity of heteromeric channels produced by coexpression of hCNGA3 with hCNGB3 mutants (S435F and D633G) to blockade by extracellular Ca2 was tested in a similar way.
The current ratios at 80 and 80 mV recorded in the presence of 1.8 mM Ca2 for hCNGA3/hCNGB3-S435F (Fig.
(C, D) IV relationships for hCNGA3/hCNGB3-S435F (C) and hCNGA3/ hCNGB3-D633G (D) heteromeric channels in the presence of extrac 1ellular Ca2 at concentrations of 1 10-10 (filled circle) and 1.8 0-3 M (open circle).
(E) Current ratios obtained by normalizing the amplitude at 80 mV with reference to that at 80 mV for hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3D633G channels.
*P 0.05 and **P 0.01 when current ratio for hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, or hCNGA3/hCNGB3D633G is compared with that for hCNGA3.
The Myc-tagged hCNGA3 subunit associated with the coexpressed FLAG-tagged hCNGB3, hCNGB3-S435F, and hCNGB3D633G subunits.
Furthermore, the present result demonstrated that rod monochromacy-associated S435F and D633G mutations in hCNGB3 subunit do not largely affect the sensitivity to block by extracellular Ca2 in heteromeric channels (Fig.
In summary, the present study provided the functional evidence to suggest that coexpression of hCNGA3 with rod monochromacy-associated mutants of hCNGB3 (hCNGB3S435F and hCNGB3-D633G) results in a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to the pathogenesis of the disease.
D633T
protein
substitution
true positive
Q9NQW8
Moreover, the apparent affinity for cGMP (24.8 1.7 M, n 4) in hCNGA3/hCNGB3-D633T heteromeric channels was comparable to that for wild-type hCNGA3/hCNGB3 heteromeric channels (data not shown, authors' unpublished observations, 2003).
D633G
protein
substitution
true positive
Q9NQW8
The purpose was to examine the functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G).
Macroscopic patch currents were recorded from human embryonic kidney (HEK) 293 cells expressing homomeric (hCNGA3 and hCNGB3) and heteromeric (hCNGA3/ hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3D633G) channels using inside-out patch-clamp technique.
The selectivity of hCNGA3, hCNGA3/ hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3D633G channels for monovalent cations were largely similar.
The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at From the Departments of 1Ophthalmology, 2Medical Biochemistry, and 3Physiology, Shiga University of Medical Science, Seta, Otsu, Japan; and 4Institute of Vision Research, Nagoya, Japan.
We have not detected any of the previously reported mutations but have found one novel missense mutation in hCNGB3 gene D633G, which resides in cyclic nucleotide-binding domain near the COOH terminus in CNG channel -subunit (Okada A, IOVS 2001;42:ARVO Abstract 3432).
Furthermore, we characterized the functional consequences of rod monochromacy-associated S435F6,12 and D633G mutations in hCNGB3 in a similar way.
Two mutations, 1304C T (S435F) and 1868A G (D633G), designated hCNGB3-S435F and hCNGB3-D633G, respectively, were each introduced to the hCNGB3 subunit cDNA as previously described,16 and these mutant cDNAs were also cloned into pCR3.1.
The amounts of each vector were ( g/ dish): 1.5 hCNGA3 and 0.5 GFP or 0.75 hCNGA3, 0.75 hCNGB3 (or hCNGB3-S435F, hCNGB3-D633G) and 0.5 GFP.
The activation of hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3S435F, and hCNGA3/hCNGB3-D633G channels typically exhibited little if any rundown or runup phenomena during exposure to various concentrations (2100 M) of cGMP for a period of at least 20 minutes.
The hCNGB3, hCNGB3S435F, and hCNGB3-D633G subunit cDNAs were cloned into the KpnI site of pFLAG-CMV plasmid (Sigma Chemical Co., St.
7 Rod Monochromacy-Associated hCNGB3 Mutations 2327 Effects of Rod Monochromacy-Associated Mutations in hCNGB3 on Apparent Affinity for cGMP in Heteromeric Channels The cGMP sensitivity of the heteromeric channels composed of hCNGA3 and rod monochromacy-associated mutants of hCNGB3 (hCNGB3-D633G and hCNGB3-S435F) was characterized to clarify the functional consequences of these two mutations in the hCNGB3.
Application of cGMP concentrationdependently increased the macroscopic patch currents that exhibited an essentially linear IV relationship with a reversal potential of 0 mV in HEK293 cells expressing hCNGA3/ hCNGB3-D633G (Fig.
Figures 4C and 4D, respectively, represent the concentrationresponse relationships for the activation of hCNGA3/hCNGB3-D633G and hCNGA3/hCNGB3S435F heteromeric channels by cGMP.
The solid curves through the data points represent least-squares fit of the Hill equation, yielding K1/2 of 11.9 1.0 M and nH of 1.87 0.15 for hCNGA3/hCNGB3-D633G (n 11; Fig.
Figure 5 illustrates representative IV relationships for hCNGA3 (A), hCNGA3/ hCNGB3 (B), hCNGA3/hCNGB3-D633G (C), and hCNGA3/ hCNGB3-S435F (D) channels activated by 100 M cGMP under the various bi-ionic solutions.
Macropatch currents activated by various concentrations of cGMP, recorded from HEK293 cells expressing hCNGA3/hCNGB3D633G (A) and hCNGA3/hCNGB3-S435F (B) heteromeric channels.
Concentrationresponse relationships for the activation of hCNGA3/ hCNGB3-D633G (C, filled squares) and hCNGA3/hCNGB3-S435F (D, open squares) heteromeric channels.
The K1/2 value for the activation of both hCNGA3/hCNGB3-D633G and hCNGA3/hCNGB3-S435F is thus similar to that for hCNGA3 homomers (11.1 1.0 M) but is significantly smaller than that for hCNGA3/hCNGB3 heteromers (26.2 1.9 M, P 0.001).
Immunoprecipitation experiments showed that both mutants of hCNGB3 (hCNGB3-D633G and hCNGB3-S435F) were associated with the hCNGA3 subunit (Fig.
Panels A, B, C, and D represent the current traces recorded from hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-D633G, and hCNGA3/hCNGB3-S435F channels, respectively.
Relative Permeability and Conductance Ratios of Wild-Type and Mutant Channels for Monovalent Cations Relative Permeability Ratio (PC/PK) h Relative Conductance Ratio (GC/GK) C K 1.0 1.0 1.0 1.0 N a 0.01 0.01 0.01 0.02 L i 0.02 0.03 0.08 0.04 R b 0.02 0.02 0.04 0.03 s 0.03 0.05 0.11 0.03 K Na 1.70 1.12 1.43 1.84 0.06 0.06 0.09 0.04 L i 0.01 0.01 0.01 0.08 R b 0.02 0.02 0.04 0.04 C s 0.01 0.03 0.06 0.06 CNGA3 hCNGA3/hCNGB3 hCNGA3/hCNGB3-D633G hCNGA3/hCNGB3-S435F Data are mean values 0.96 1.00 0.97 0.98 0.76 0.88 0.75 0.77 0.84 0.83 0.87 0.77 0.51 0.61 0.51 0.46 1.0 1.0 1.0 1.0 0.57 0.57 0.56 0.60 0.38 0.58 0.51 0.36 0.27 0.40 0.28 0.23 SEM from 4 8 patches.
(hCNGA3) and heteromeric (hCNGA3/hCNGB3, hCNGA3/ hCNGB3-D633G and hCNGA3/hCNGB3-S435F) channels were not appreciably affected (data not shown).
The present study further characterized the functional consequences of the rod monochromacy-associated mutations in hCNGB3 (S435F and D633G).
However, the heteromeric channels composed of hCNGA3 and rod monochromacy-associated mutants of hCNGB3 (hCNGB3-D633G and hCNGB3-S435F) exhibited a higher apparent affinity for cGMP compared with wild-type heteromeric channels (Fig.
This functional alteration in cone CNG channels associated with D633G or S435F mutation in hCNGB3 should cause the channels to be reluctant to the decrease in intracellular cGMP level after reception of photons, leading to their being always open, which should be responsible at least partly for the pathogenesis of rod monochromacy.
The sensitivity of heteromeric channels produced by coexpression of hCNGA3 with hCNGB3 mutants (S435F and D633G) to blockade by extracellular Ca2 was tested in a similar way.
6C) and hCNGA3/hCNGB3-D633G (Fig.
The present study also detected an increase in the apparent affinity for cGMP in heteromeric cone CNG channels containing the rod monochromacy-associated D633G mutation in hCNGB3 subunits (Fig.
(C, D) IV relationships for hCNGA3/hCNGB3-S435F (C) and hCNGA3/ hCNGB3-D633G (D) heteromeric channels in the presence of extrac 1ellular Ca2 at concentrations of 1 10-10 (filled circle) and 1.8 0-3 M (open circle).
(E) Current ratios obtained by normalizing the amplitude at 80 mV with reference to that at 80 mV for hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3D633G channels.
*P 0.05 and **P 0.01 when current ratio for hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, or hCNGA3/hCNGB3D633G is compared with that for hCNGA3.
The Myc-tagged hCNGA3 subunit associated with the coexpressed FLAG-tagged hCNGB3, hCNGB3-S435F, and hCNGB3D633G subunits.
Furthermore, the present result demonstrated that rod monochromacy-associated S435F and D633G mutations in hCNGB3 subunit do not largely affect the sensitivity to block by extracellular Ca2 in heteromeric channels (Fig.
In summary, the present study provided the functional evidence to suggest that coexpression of hCNGA3 with rod monochromacy-associated mutants of hCNGB3 (hCNGB3S435F and hCNGB3-D633G) results in a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to the pathogenesis of the disease.
S435A
protein
substitution
true positive
Q9NQW8
It is likely that phenylalanine with bulky aromatic side chain at this position destabilizes the closed state of the channel and is thereby responsible for the increase in the cGMP sensitivity.26 When coexpressed with hCNGA3, the S435A mutant of hCNGB3 subunit produceed functional heteromeric channels with the FIGURE 7.
11274446
full text
I176C
protein
substitution
P63252
true positive
1), as shown for the c orresponding I176C residue in K ir2.1 (10).
Moreover, as w ith K ir2.1[I176C], modification of indiv idual residues leads to only a partial reduction of i.
A sequence Kir2.1[I176C] (10)].
L164C
protein
substitution
true positive
Q14654
2-Aminoethyl methanethiosulfonate (MTSEA ) failed to modify Cd2 -insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels.
Although a single Cd2 ion is coordinated by L164C, four MTSEA ``hits'' can occur, each sequentially reducing the single-channel current.
A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd2 -insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA modification of the dimer occurs in two hits.
L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr ), even though qBBr caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA ), implying that qBBr can also enter the inner cavity but does not modify L164C residues.
A stable conformation optimally places the four L164C side chains for coordination of a single Cd2 ion.
Residue 164 in K ir6.2 is equivalent to residue 176 in K ir2.1, and in KAT P channels generated by K ir6.2[L164C] mut ant subun its, a single Cd2 ion was c oordinated by at least three c ysteines, indicating a narrow diameter ( 6 ) at the level of L164C (11).
I n K ir6.2[L164C] mut ant channels, we now demonstrate that ala though a single Cd2 ion is c oordinated, four ``hits'' by MTSEA nd t wo or three by MTSP TrEA can also oc cur.
Derivatizations of L164C by MTSEA , MTSP TrEA , and qBBr were processed w ith biopoly mer f rom the st arting K ir6.2 model.
Cd2 and MTSEA modification of L164C residues (A) Effects of Cd2 nd MTSEA on representative inside-out patch currents from COSm6 cells cotransfected with SUR1 and Control-Kir6.2 (Control, Top), Kir6.2[L164C] (L164C, Middle), or control-Kir6.2-G6-Kir6.2[L164C] (control-L164C dimer, Bottom).
4 C and D), inward currents are shown as upward deflections, zero current is indicated by a dashed line, and cartoons indicate the tetrameric makeup of L164C (F) and wild-type 164L (E) subunits.
K ir6.2[L164C] mut ant channels (L164C) are strongly and only 30 s; see Fig.
The open probabilit y of L164C mut ant channels is ver y high ( 0.9), and only a single short-lived closed time is obser ved [ 0.5 ms (17)].
mM MTSEA MTSEA Blocks One Cd2 Is Sufficient to Inhibit the Channel by Interaction with Three or Four L164C Residues.
Ex pression of c ontrol-K ir6.2 SUR1 gen- L164C Channels in Four Hits.
Control L164C Dimer Has No High-Affinity Cd2 Block but Is Inhibited by MTSEA .
To gain further insight to the nature of the Cd2 and MTSEA interactions w ith L164C, we c onstr ucted a dimeric fusion (c ontrol L164C) of c ontrol-K ir6.2 and L164C subun its.
Unlike homomeric L164C channels, both macrosc opic (Fig.
Multiple hits by MTSEA at residue L164C.
(A) Representative single SUR1 L164C channel in inside-out patch.
(B and C) (Upper) Representative single SUR1 L164C (A) and SUR1 Control-L164C dimeric (B) channels in inside-out patch, after exposure to 0.1 mM MTSEA .
(D) Conductance levels observed after MTSEA its (solid symbols) as a percentage of full conductance (open symbols) for individual patches of five homomeric L164C channels and three dimeric Control-L164C channels.
* indicates a L164C channel expressed without SUR1.
The above dat a indicate that multiple MTSEA hits are possible, even though a single Cd2 ion is c oordinated in L164C channels.
In macrosc opic L164C patches, rec over y f rom Cd2 inhibition t akes place w i t h rec 30 s (Fig.
Ac c ordingly, we attempted to modif y L164C w ith the use of uncharged methyl methanethiosulfonate (MMTS).
There is a small reduction of macrosc opic current in L164C (Fig.
, but Not qBBr , Modifies Kir6.2[L164C].
Coordination of a single Cd2 ion at L164C requires that the pore be 6- diameter at this point, although the dat a above clearly show that four MTSEA moieties can modif y these same residues.
As discussed below, modification of all four L164C residues by MTSEA is actually sterically feasible, even in such a narrow pore (11, 13), and requires no movement of backbone M2 helices.
4 (A and B) shows original rec ords and averaged dat a f rom c ontrol-K ir6.2, L164C, and dimeric channels ex posed to 5 mM MTSP TrEA .
There was no sign ificant modification of c ontrol-K ir6.2 by this agent, but 95% modification of L164C.
This level of inhibition is sign ificantly less modification than what oc curs in the homomeric L164C channel, indicating that at least t wo c ysteines can be modified in the latter [t wo c ysteines are apparently modified by MTSP TrEA in MTSPTrEA rapidly and irreversibly reduces L164C mut ant channel current by 90% (Fig.
Af ter ex posure of a single L164C channel to MTSEA , up to four hits oc cur (Fig.
In the c ontrol L164C dimer, Loussouarn et al.
(A) Macroscopic current recorded from an inside-out SUR1 L164C patch.
(C) Representative record of single SUR1 L164C channel in inside-out patch.
(E) Effect of MMTS on representative inside-out patch currents from cells cotransfected with SUR1 and either Control-Kir6.2 (Control, Left) or L164C (Right).
(G) Effect of MMTS on Cd2 block of representative L164C inside-out patch current.
MTSPTrEA , but not qBBr , modifies L164C.
(A) Effect of MTSPTrEA n representative inside-out patch currents from cells cotransfected with SUR1 and Control-Kir6.2 (Control, Top), L164C (Middle), or Control-L164C dimer (Bottom).
(C) Effect of qBBr on representative L164C inside-out patch current.
(10), there was no modification of L164C channels by 5 mM qBBr (Fig.
Four MTSEA derivatives are attached to mutant T107C (equivd alent to L164C in Kir6.2).
(B) Transparent representations of the water-accessible pore of Kir6.2, with space-filling representations of MTS derivatives attached to L164C as indicated.
Three K ions and one water molecule are indicated in the c selectivity filter, and a Cd2 ion is coordinated by residues L164C.
(Lower) Cd2 oordinated by four L164C-S sulfhydryls, viewed from beneath the membrane.
The side chains of residue L164C (indicated) are optimally positioned in the model structure for coordination of Cd2 .
To examine the likelihood of Cd2 c oordination by L164C, a homotetrameric model of K ir6.2[L164C] f ragment 78 176 was built w ith a Cd2 ion.
The L164C side chains, near the bottom of the cav it y, are pointing to the central ax is and c oordinate to the Cd2 ion, as is more clearly v isible in the 90 projection (Fig.
7 4231 PHYSIOLOGY Kir6.2[L164C] (Fig.
Even MTSP TrEA modification reduces the c onduct ance of homomeric L164C channels almost c ompletely but reduces the current through dimeric channels by only 70%, indicating that at least t wo moieties can simult aneously oc cupy the K ir6.2 pore.
A lthough we see no ev idence of qBBr modification of L164C channels, this reagent does cause volt age-dependent pore block, indicating that at least one qBBr molecule can also enter the pore but may be sterically restricted f rom modif ying the 164C residue.
T107C
protein
substitution
P0A333
true positive
Four MTSEA derivatives are attached to mutant T107C (equivd alent to L164C in Kir6.2).
6B, Left) or the equivalent KcsA[T107C] (Fig.
C166S
protein
substitution
true positive
Q14654
The ``c ontrol'' K ir6.2 c onstr uct had a deletion of 36 amino acids f rom the C-ter minal end, as well as N160D and C166S mut ations (11).
N160D
protein
substitution
true positive
Q14654
The ``c ontrol'' K ir6.2 c onstr uct had a deletion of 36 amino acids f rom the C-ter minal end, as well as N160D and C166S mut ations (11).
C164S
protein
substitution
true negative
The dist ance bet ween t wo C164-S is about 5 , too long to allow a disulfide bridge, but per fect for binding a cadmium ion (the S -Cd2 dist ance is about 2.55 ).
We used t wo template models for c oordination of Cd2 : a tetrahedral organ ization of the four C164-S coordinated to one central Cd2 (Fig.
tion of four equatorial C164-S and t wo added ax ial water molecules c oordinate to one central Cd2 (Fig.
12654924
full text
E1337K
protein
substitution
Q02294
true positive
Clone 5M had the following amino acid substitutions: Q1327K, D1330L, E1334N, E1337K, and Q1339R inserted by PCR.
Q1327K, D1330L, E1334N, E1337K, and Q1339R for the quintuple mutant plus an additional substitution of E1332R in the sextuple mutant).
Q1339R
protein
substitution
Q02294
true positive
Clone 5M had the following amino acid substitutions: Q1327K, D1330L, E1334N, E1337K, and Q1339R inserted by PCR.
Q1327K, D1330L, E1334N, E1337K, and Q1339R for the quintuple mutant plus an additional substitution of E1332R in the sextuple mutant).
G1326P
protein
substitution
Q02294
true positive
We also added the G1326P mutation (which by MVIIA Block of N-type Channels 20173 FIG.
E1332R
protein
substitution
Q02294
true positive
Clone 6M1 had an additional substitution, E1332R, in combination with the five aforementioned mutations.
We next wanted to determine whether the effects of these five mutations were additive to a separate point mutation, E1332R, which was found previously to slow the time constant of development of the block (7).
Q1327K, D1330L, E1334N, E1337K, and Q1339R for the quintuple mutant plus an additional substitution of E1332R in the sextuple mutant).
E1334N
protein
substitution
Q02294
true positive
Clone 5M had the following amino acid substitutions: Q1327K, D1330L, E1334N, E1337K, and Q1339R inserted by PCR.
Q1327K, D1330L, E1334N, E1337K, and Q1339R for the quintuple mutant plus an additional substitution of E1332R in the sextuple mutant).
D1330L
protein
substitution
Q02294
true positive
Clone 5M had the following amino acid substitutions: Q1327K, D1330L, E1334N, E1337K, and Q1339R inserted by PCR.
Q1327K, D1330L, E1334N, E1337K, and Q1339R for the quintuple mutant plus an additional substitution of E1332R in the sextuple mutant).
Q1327K
protein
substitution
Q02294
true positive
Clone 5M had the following amino acid substitutions: Q1327K, D1330L, E1334N, E1337K, and Q1339R inserted by PCR.
Q1327K, D1330L, E1334N, E1337K, and Q1339R for the quintuple mutant plus an additional substitution of E1332R in the sextuple mutant).
14734807
full text
E28K
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
The reduced glycerol per meabilit y obser ved in E28K, charge inversion, E28A, smaller side chain, and T31V hydrophobicit y increase, was st atistically sign ificant, and the ef fect T31A was st atistically borderline (Table 1).
S78W
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
T31V
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
The reduced glycerol per meabilit y obser ved in E28K, charge inversion, E28A, smaller side chain, and T31V hydrophobicit y increase, was st atistically sign ificant, and the ef fect T31A was st atistically borderline (Table 1).
T31A
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
The reduced glycerol per meabilit y obser ved in E28K, charge inversion, E28A, smaller side chain, and T31V hydrophobicit y increase, was st atistically sign ificant, and the ef fect T31A was st atistically borderline (Table 1).
E28A
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
The reduced glycerol per meabilit y obser ved in E28K, charge inversion, E28A, smaller side chain, and T31V hydrophobicit y increase, was st atistically sign ificant, and the ef fect T31A was st atistically borderline (Table 1).
S200P
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
E125S
protein
substitution
true negative
Finally, the ef fect of the E125S mut ation on the Arrhen ius activation energ y was measured in c omparison to the w ild-t ype channel and c ontrol ooc y tes (Fig.
Arrhenius plot of the water permeability (Pf) through PfAQP wildtype (circles), PfAQP E125S (squares), and control oocyte membranes (triangles).
The difference in activation energies of the E125S mut ant and w ild-t ype PNAS February 3, 2004 vol.
This is ev ident f rom the increase of the activation energ y in the PfAQP E125S mut ant f rom 3.5 kcal mol (PfAQP w ild-t ype) to 7.5 kcal mol and, c onsequently, 10-fold reduction in water per meabilit y.
L192V
protein
substitution
true negative
Mut ant L192M was barely ex pressed, whereas the L192V c onstr uct was present at PfAQP w ild-t ype levels.
Nevertheless, the high per meabilit y of the L192V mut ant for both water and glycerol indicated that L192(M202) probably does c ontribute little to discriminate bet ween water and glycerol c onduct ance in PfAQP.
(B) Permeability of pentitols through PfAQP and the L192V mutant (n 35).
PfAQP L192V mut ant sign ificantly decreased the per meabilit y for x ylitol (P 0.01) and D-arabitol (P 0.02) and raised that of ribitol 3-fold (P 0.05; Fig.
E28V
protein
substitution
true negative
In Mutation None (WT) E28V E28K E28A T31V T31A E28A T31A S78W S200P Location TM1 Water glycerol 1.43 1.68 1.08* 0.84* 0.99* 0.99 1.21 1.35 1.21 TM1 Loop B Loop E TM, transmembrane.
C190S
protein
substitution
true negative
A mercur y-insensitive mut ant AQP1 C190S was c oinjected when the mercur y inhibition of PfAQP mut ants was a s s a y e d .
W124C
protein
substitution
true negative
To further prove that the WET triad of the PfAQP C-loop is closely c onnected w ith the pore function, we mut ated the tr yptophan to c ysteine (W124C).
PfAQP W124C was ex pressed in Xenopus ooc y tes (Fig.
(B) Effect of Hg2 on water and glycerol permeation of PfAQP W124C.
L192M
protein
substitution
true negative
Mut ant L192M was barely ex pressed, whereas the L192V c onstr uct was present at PfAQP w ild-t ype levels.
The L192M mut ant was poorly ex pressed, and increased water and glycerol per meabilit y was ex pressed only moderately c ompared to c ontrol injected ooc y tes (Fig.
In agreement w ith the reduction of water per meabilit y by L192M, this assigns this amino acid a defin itive, albeit possibly minor, role in c ontrolling PfAQP solute per meabilit y.
10477148
full text
L43P
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
V206D
protein
substitution
true negative
In vitro expression of other mutations in this loop (R181C, T204N, Y205C, V206D) has shown that ligand binding is not totally abolished (18 20).
R181C
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
In vitro expression of other mutations in this loop (R181C, T204N, Y205C, V206D) has shown that ligand binding is not totally abolished (18 20).
In our experience, however, the R181C, T204N, and Y205C mutations cause the complete clinical phenotype (Table 2).
T204N
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
In vitro expression of other mutations in this loop (R181C, T204N, Y205C, V206D) has shown that ligand binding is not totally abolished (18 20).
In our experience, however, the R181C, T204N, and Y205C mutations cause the complete clinical phenotype (Table 2).
Administration of a double dose of DDAVP to our patient with the T204N mutation, however, did result in a rise of urine osmolality from 126 to 230 mosmol/kg (18), suggesting that, at least in nonphysiologic circumstances, higher values can be reached.
S216P
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
Y205C
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
In vitro expression of other mutations in this loop (R181C, T204N, Y205C, V206D) has shown that ligand binding is not totally abolished (18 20).
In our experience, however, the R181C, T204N, and Y205C mutations cause the complete clinical phenotype (Table 2).
V88M
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
After a water deprivation test, two patients reached values of 545 mosmol/kg (V2 receptor: V88M) and 1016 (V2 receptor: R337X), whereas, after rehydration, they showed no response to (DD)AVP.
L44F
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
Q225X
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
D85N
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
R337X
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
After a water deprivation test, two patients reached values of 545 mosmol/kg (V2 receptor: V88M) and 1016 (V2 receptor: R337X), whereas, after rehydration, they showed no response to (DD)AVP.
In two of 30 patients, both with a V2 receptor defect (g(intron B)a and R337X resp.), severe dilation of the urinary tract occurred (Figure 2).
R187C
protein
substitution
true negative
Dehydration was mentioned as the cause of death in five young children from two different families, one with a V2 receptor (large deletion of the 5 region) and one with an AQP2 defect (R187C).
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
L62P
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
G185C
protein
substitution
true negative
Except for a possibly milder phenotype in patients with a G185C mutation, no clear relationship between clinical and genetic data could be found.
One of these family members had been mistaken for a case of central diabetes insipidus in the past (V2 receptor: G185C).
In one of these patients, the elder family members had not been diagnosed until the present study (V2 receptor: G185C).
Three older patients (ages 9, 10, and 17 yr, respectively), all with the V2 receptor mutation G185C, were referred for reasons that were not related or were indirectly related to NDI.
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
The other two patients with a G185C mutation are younger family members, which might explain why they were diagnosed earlier.
Considering maximal urine osmolality and age at diagnosis, the only mutation in our study group with a putative milder phenotype could be the G185C mutation.
Yet, since the G185C mutation is located in the second extracellular loop of the V2 receptor, which is thought to be the AVP binding region, this mutation might cause partial NDI.
Thus, despite the difficult interpretation of clinical data, the G185C mutation might be an interesting candidate for in vitro expression studies.
S167L
protein
substitution
true negative
These patients shared a glycine 185 to cysteine (G185C) amino Maximum Urine Osmolality (u.o., mosmol/kg) or Specific Gravity (s.g.) Comments V2 receptor gene del complete gene del complete gene del 5 region del 5 region del 5 region L43P L44F L62P L62P D85N D85N V88M 528del7 S167L S167L R181C G185C G185C G185C G185C G185C T204N Y205C Q225X g(intron B)a 977(978)delG R337X R337X AQP2 gene R187C/S216P R187C 3.5 0.5 2.5 8.5 0.5 7 6.8 53 21 32 32 16 2.5 28 8 6 128 108 9 204 7 15 2.5 8.5 3 12 16 4 198 (u.o.) 84 (u.o.) 1000 (s.g.) 95 (u.o.) 93 (u.o.) 1004 (s.g.) 1002 (s.g.) 90 (u.o.) After DDAVP After DDAVP With serum Na 158 mmol/L AVP no effect After DDAVP With s.o.
11886861
full text
T248V
protein
substitution
true positive
O54912
Grouped comparisons combining all TASK-1 constructs indicated that both effects on this mutant were smaller than those on the wild-type channel (and on the C-terminal deletion and T248V mutants) and greater than the effects on the two TREK-1 substitution mutants.
R245W
protein
substitution
true positive
O54912
C and D, mean data show statistically significant effects of R245W mutation (as determined by one-way ANOVA between the three groups; n 6 for each group); asterisks indicate difference from wild-type (WT) TASK-1 (p 0.05).
14715535
full text
D307H
protein
substitution
true negative
In this study, we explored the effect of adenoviral-directed expression of a canine CASQ2 protein carrying the catecholaminergic polymorphic ventricular tachycardialinked mutation D307H (CASQ2D307H) on Ca2 signaling in adult rat myocytes.
Total CASQ2 protein levels were consistently elevated 4-fold in cells infected with adenoviruses expressing either wild-type CASQ2 (CASQ2WT) or CASQ2D307H.
Expression of CASQ2D307H reduced the Ca2 storing capacity of the SR.
In addition, the amplitude, duration, and rise time of macroscopic ICa-induced Ca2 transients and of spontaneous Ca2 sparks were reduced significantly in myocytes expressing CASQ2D307H.
Myocytes expressing CASQ2D307H also displayed drastic disturbances of rhythmic oscillations in [Ca2 ]i and membrane potential, with signs of delayed afterdepolarizations when undergoing periodic pacing and exposed to isoproterenol.
Our data suggest that the arrhythmogenic CASQ2D307H mutation impairs SR Ca2 b toring and release functions and destabilizes the Ca2 -induced Ca2 release mechanism by reducing the effective Ca2 uffering inside the SR and/or by altering the responsiveness of the Ca2 release channel complex to luminal Ca2 .
regulation of SR Ca2 release by controlling the local luminal [Ca2 ] in the vicinity of the RyR2 channels14 and possibly also by serving as a luminal Ca2 sensor for RyR2.15 The recessive form of CPVT was positionally mapped in several Bedouin families to the region of chromosome 1 (1p 13-21) in which the CASQ2 gene is located.16 Subsequent sequence analysis of CASQ2 genes from these individuals identified a missense point mutation in a highly conserved region of CASQ2.5 This mutation (referred to here as CASQ2D307H) converts a negatively charged aspartic acid into a histidine in a putative Ca2 chelating region of CASQ2.
Lahat et al5 proposed that this mutation exerts its effects by disrupting Ca2 binding to CASQ2, but the specific mechanisms whereby the D307H mutation causes CPVT remain unknown.
In the present study, we used an adenoviralmediated gene transfer strategy to express a canine CASQ2D307H protein in cardiac myocytes and explored the effects of this mutation on intracellular Ca2 handling using Ca2 imaging and patch-clamp techniques.
Our results establish a pathological link between the expression of CASQ2D307H and the clinical phenotype observed in patients carrying this mutation.
Materials and Methods Construction of Recombinant Adenoviruses The construction of Ad-CSQ2WT and Ad-Control was described previously.14 The D307H mutation was introduced into the fulllength canine CASQ2 cDNA using the Quikchange Site-Directed Mutagenesis Kit (Stratagene).
The CASQ2D307H coding region was transferred into the Adeno-X Viral DNA, and recombinant adenoviruses were generated according to the instructions of the manufacturer (Clontech).
Results Adenoviral-Mediated Expression of Mutant CASQ2 in Isolated Rat Myocytes The dog and human CASQ2 proteins display 91% sequence identity overall, and the D307H mutation is located in a region that is highly conserved among CASQ2 orthologues from various vertebrate species (Figures 1A and 1B).
The D307H mutation was introduced into the coding sequence of canine CASQ2 by site-directed mutagenesis.
The canine CASQ2D307H coding sequence was inserted into an adenoviral vector to generate Ad-CASQ2D307H for subsequent gene transfer into adult rat ventricular myocytes.
Infection of myocytes with either Ad-CASQ2WT or AdCASQ2D307H consistently resulted in equivalent 4-fold increases in total CASQ2 protein, and CASQ2 levels were unchanged in cells infected with Ad-Control.
In these experiments the pipette solution contained K -aspartate CASQ2D307H Overexpression Decreases the SR Ca2 Storage Capacity Caffeine applications (10 mmol/L) were used to assess the changes in the total SR Ca2 content in each group of r adenovirus-infected myocytes.
B, Comparison of sequence around the D307H mutation in CASQ2 and the related CASQ1 proteins.
C, Western analysis of CASQ2 protein levels in cardiac myocytes infected with the control adenovirus or adenoviruses expressing wild-type CASQ2 or CASQ2D307H.
A, Caffeine-induced [Ca2 ]i transients (upper traces) and INCX (lower traces) in myocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors.
The integral of INCX was 2.3-fold higher in Ad-CASQ2WT myocytes and decreased to 36% of control in Ad-CASQ2D307H myocytes (Figure 2C).
Thus, ectopic expression of CASQ2D307H suppressed the ability of SR to store Ca2 .
In contrast, expression of CASQ2D307H reduced the amplitude of the caffeine-induced fluorescence signal to 41% of control (Figure 2B).
These changes in fluorescence signals in Ad-CASQ2WT and Ad-CASQ2D307H myocytes were paralTABLE 1.
Parameters of ICa and Ca2 IC a Peak Amplitude, pA Ad-Control Ad-CASQ2WT Ad-CASQ2 D307H The effects of expressing CASQ2WT and CASQ2D307H on ICa and intracellular [Ca2 ] transients in patch-clamped myocytes are illustrated in Figure 3.
There were no apparent changes in the parameters of ICa in myocytes expressing either CASQ2WT or CASQ2D307H (Figure 3A, bottom, and Figure 3B; Table 1).
Thus, expression of CASQ2WT or t CASQ2D307H did not change the characteristics of the Ca2 rigger for Ca2 release from the SR.
A, Representative line-scan images of Ca2 sparks acquired in myocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors.
B, Surface plots of averaged Ca2 sparks in myocytes infected with Ad-Control (30 events), Ad-CASQ2WT (24 events), and Ad-CASQ2D307H (29 events) vectors.
A, Traces of ICa (lower traces) and Ca2 transients (upper traces) induced by depolarization from 50 to 0 mV in cardiomyocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors.
B and C, Voltage dependence of ICa (B) and Ca2 transients (C) in myocytes infected with Ad-Control, Ad-CASQ2WT, and Ad-CASQ2D307H vectors (n for each point ranged from 5 to 18).
Importantly, the duration of the rising phase was increased by 90%, consistent with the hypothesis that CASQ2 modulates SR Ca2 release by prolonging the duration of the Ca2 flux from the SR to the cytosol.14 In contrast, in myocytes expressing CASQ2D307H, the Ca2 transients were drastically reduced in size and duration.
Furthermore, the rise time of Ca2 transients in Ad-CASQ2D307H myocytes was shortened significantly compared with control (by 39%).
Thus, expressing CASQ2D307H inhibited active SR Ca2 release, apparently by shortening Ca2 release duration.
Ca2 S parks in Permeabilized Myocytes We next examined the impact of expressing CASQ2D307H on properties of focal fluorescence signals, ie, Ca2 sparks, in permeabilized myocytes.
Myocytes were permeabilized with saponin, and Ca2 sparks were recorded at a constant cytosolic [Ca2 ] of 100 nmol/L.18 Representative line-scan images of sparks acquired in myocytes infected with AdControl, Ad-CASQ2WT, and Ad-CASQ2D307H are shown in Figure 4A, and surface plots of sparks obtained by averaging multiple individual events12 acquired in the same three groups of myocytes are illustrated in Figure 4B.
The impact of expression of CASQ2WT and CASQ2D307H on parameters of Ca2 sparks is documented in Table 2.
However, when CASQ2D307H was expressed, the Ca2 sparks were smaller and briefer and had rise times shorter than in control (73% of control).
Thus, expressing CASQ2D307H resulted in focal release events of reduced size and abbreviated duration.
The effects of isoproterenol (ISO) treatment (1 mol/L) on periodic Ca2 transients in control myocytes and in myocytes expressing either CASQ2WT or CASQ2D307H is illustrated in Figure 5.
In myocytes expressing CASQ2D307H, the amplitude of Ca2 transients was reduced with respect to control, consistent with measurements in resting c myocytes.
These results indicate that r expression of CASQ2D307H not only reduces the amount of Ca2 eleased from the SR but also destabilizes the Ca2 release mechanism, leading to spontaneous, premature discharges of SR Ca2 stores in myocytes undergoing periodic pacing.
Parameters of Ca2 F/F0 Ad-Control Ad-CASQ2WT Ad-CASQ2D307H 1.62 0.02 2.26 0.04* 1.46 0.02* S Ca2 C ycling in Rhythmically Paced Myocytes parks DHA, ms 14.9 0.2 24.3 0.3* 11.4 0.2* FWHM, m 2.45 0.02 3.07 0.03* 1.93 0.02* Rise Time, ms 7.9 0.1 13.6 0.2* 6.2 0.1* *P 0.01 vs control (n 1414 to 1525).
Again, application of ISO produced characteristic disturbances in the periodic Ca2 transients and MP in CASQ2D307H-expressing myocytes (Figure 6, left and middle).
The objective of the present study was to investigate the functional characteristics of the missense mutation D307H identified in the first large pedigree affected by CPVT linked to mutations in CASQ2.5 Adenoviral-mediated expression of CASQ2D307H diminished the Ca2 storing and releasing capabilities of the SR in rat ventricular myocytes, thus resulting in pronounced dominant-negative effects on SR Ca2 handling.
Relevant to the arrhythmogenic consequence of the D307H mutation, our data demonstrated that myocytes expressing CASQ2D307H develop abnormal intracellular [Ca2 ] oscillations that cause DADs specifically during -adrenergic stimulation.
Disturbances in rhythmic [Ca2 ]i transients and membrane potential induced by ISO in myocytes expressing CASQ2D307H.
Recordings of membrane potential (upper traces), along with line-scan images (middle) and time-dependent profiles of [Ca2 ]i (lower traces) in myocytes infected with Ad-Control (A), Ad-CASQ2WT (B), and Ad-CASQ2D307H (C) vectors before and after exposure of the myocytes to 1 mol/L ISO.
Restoration of Normal Periodic Ca2 Transients by Increasing SR Ca2 Buffering Capacity With Low-Affinity Ca2 Buffers We have recently proposed that CASQ2 regulates the functional size and stability of SR Ca2 stores by serving as a buffer for Ca2 in the SR lumen.14 To test the hypothesis that disruption of Ca2 cycling observed in cells expressing CASQ2D307H is attributable to abnormal intra-SR Ca2 buffering, we carried out experiments using the low-affinity exogenous Ca2 buffer, citrate.
Citrate was loaded into the SR of CASQ2D307H-expressing myocytes by dialyzing them with a citrate-containing pipette solution.12 Sequestration of citrate into the SR occurs substantially slower (20 to 30 minutes) Effects of D307H on CASQ2 Function Based on the analysis of the amino acid sequence and crystal structure of calsequestrin, Asp307, which harbors this arrhythmogenic mutation, is localized to a putative Ca2 binding region between the second and third thioredoxin-like domains Figure 6.
Restoration of normal rhythmic activity by intracellular dialysis with citrate in myocytes expressing CASQ2D307H.
Recordings of membrane potential (upper traces), along with line-scan images (middle) and time-dependent profiles of [Ca2 ]i (lower traces), in myocyte infected with Ad-CASQ2D307H vector before and after exposure of the myocyte to 1 mol/L ISO.
476 Circulation Research March 5, 2004 however, that the clinical phenotype of D307H is influenced by various adaptive changes in cellular Ca2 handling mechb anisms such as increased expression of other luminal Ca2 inding proteins (eg, calreticulin) or CASQ1 isoform transition.
of the protein.5,21 Consequently, it has been hypothesized that the pathology of CPVT may involve disrupted Ca2 binding by CASQ2.5 Our finding that expression of CASQ2D307H diminished SR Ca2 releasing and storing capabilities despite the presence of normal levels of the endogenous wild-type protein suggests that the effect of this mutation on CASQ2 b function may be more complex than merely altering Ca2 inding by CASQ2 monomers.
This possibility is consistent with reduced SR Ca2 storing capacity of myocytes expressing CASQ2D307H (Figure 2).
CASQ2 has been proposed to be actively involved in regulation of Ca2 release through protein-protein interactions with RyR2, junctin, and triadin.8,24 Recent results obtained in our laboratory suggest that RyR2 complexed with junctin and triadin is inhibited by CASQ2 at low luminal [Ca2 ] and that this inhibition is relieved at high luminal [Ca2 ].15 If the D307H mutation were to affect the ability of CASQ2 to interact with the RyR2 complex, this could lead to RyR2 channels with abnormally high activity.
In this case, the diminished SR Ca2 storing and releasing functions in CASQ2D307H-expressing myocytes could reflect the compromised ability of the SR to retain Ca2 due to hyperactive, ie, leaky, RyR2 channels.
It is interesting to note that whereas the CASQ2D307H protein exerted clear dominant-negative effects in infected myocytes in our experiments, in the clinical setting, this mutation causes an abnormal phenotype only when 100% of the CASQ2 protein is abnormal (ie, in homozygous carriers of the mutation).5 This apparent discrepancy could be ascribed to relative levels of the wild-type and mutant CASQ2 proteins in our experiments, where the ratio of mutant to wild-type protein was 3:1 in myocytes infected with AdCASQ2D307H (Figure 1).
Because a 2-fold increase in total CASQ2 level did not lead to any gain in function, our results still imply that the function of CASQ2D307H should be impaired in heterozygous carriers of this mutation (50% of normal protein present).
It is likely, Molecular Mechanisms of CPVT Our results provide a plausible explanation for the cellular mechanism by which the D307H mutation of CASQ2 causes catecholaminergic tachycardia.
Exposure of CASQ2D307H myocytes to -adrenergic stimulation induced extrasystolic, spontaneous Ca2 transients and resulted in the development of DADs (Figure 5C).
The effects of expressing CASQ2D307H on specific parameters of both cellaveraged Ca2 transients and Ca2 sparks (eg, amplitude and rise-time duration; Figures 2 through 4 and Tables 1 and 2) were similar to those we observed on reducing CASQ2 protein levels.14 Thus, our previous results and present findings suggest a mechanism whereby reduced buffering of Ca2 in the SR lumen by CASQ2 (and/or disrupted interactions of CASQ2 with the RyR2 channel complex) leads to altered regulation of the Ca2 release mechanism by luminal Ca2 .
Within this mechanistic framework, the role of adrenergic stimulation in promoting the pathologic signs of disease can be ascribed to an accelerated u recharging of the SR Ca2 store (as a result of enhanced SR Ca2 ptake by the CaATPase) and hence further contributing to the premature functional restitution of the RyR2s.14 Our finding that normal Ca2 cycling in CASQ2D307H-expressing myocytes can be b restored by loading their SR with low-affinity exogenous Ca2 uffers (Figure 6) provides a strong support for the proposed role of CASQ2 and luminal Ca2 in the pathogenesis of CPVT.
Viatchenko-Karpinski et al Sudden Cardiac Death Associated With CASQ Mutation 477 Conclusions In conclusion, we have established a pathological link between the D307H mutation in the CASQ2 gene and the clinical phenotype observed in CPVT patients carrying this mutation.
Thus, characterization of the effects of CASQ2D307H in rat myocytes has elucidated the potential mechanisms by which mutations in CASQ2 determine the arrhythmia-prone substrate observed in CPVT patients.
14715536
full text
S2808D
protein
substitution
true positive
Q92736
The S2808D mutant thought to mimic constitutive phosphorylation also retained the ability to bind FKBP12.6.
Investigations of single RyR2 channels from failing hearts in lipid bilayers have revealed an R enhanced sensitivity of the channel to Ca2 activation and altered gating and conduction.8 Parallel to these observations, phosphorylation of RyR2 from normal hearts by exogenous PKA caused dissociation of FKBP12.6 from RyR2 and alterations in channel activation, gating, and conduction.8 Furthermore, a single mutation of serine-2808 to aspartate (S2808D) abolished FKBP12.6 binding to RyR2.10 These observations have led to the notions that FKBP12.6 cannot bind to serine-2808 phosphorylated RyR2 and that PKA phosphorylation physiologically regulates FKBP12.6-RyR2 interaction.8,10 As a result, alteration in FKBP12.6-RyR2 interaction attributable to PKA hyperphosphorylation was put forward as a novel mechanism for cardiac dysfunction in heart failure and exercise-induced sudden cardiac death.8,10 A central feature of the theory proposed by Marks et al6 for heart failure is the idea that PKA phosphorylation at a single site, serine-2808, in RyR2 induces dissociation of FKBP12.6.
In the present study, we have focused on investigations of whether FKBP12.6 can bind to serine-2808 phosphorylated RyR2 and the S2808D mutant and whether PKA phosphorylation at serine-2808 can dissociate FKBP12.6 from RyR2.
We have demonstrated that FKBP12.6 is able to interact with both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2 and with the S2808D mutant.
Mutations S2808A and S2808D were introduced into the mouse RyR2 by the overlap extension method.
Analysis of FKBP12.6 interaction with RyR2 or mutants S2808A and S2808D was performed by coimmunoprecipitation assay using HEK293 cell lysate or solubilized canine heart microsome.
Anti-RyR(34c) was used for immunoprecipitation and detection of RyR2, S2808A, and S2808D.
Precipitation of the RyR2(wt), S2808A, and S2808D by GST-FKBP12.6.
The expressed RyR2(wt) (lane 1), S2808A (lane 2), and S2808D (lane 3) proteins were precipitated from cell lysates by GST-FKBP12.6 or by GST alone.
On the other hand, the S2808D mutant, which is thought to mimic phosphorylation, may correspond to a constitutively phosphorylated state of serine-2808.10 If the phosphorylation state of serine-2808 dictates the interaction between FKBP12.6 and RyR2, RyR2 wt and the S2808A and S2808D mutants would be expected to differ markedly in their ability to interact with FKBP12.6.
To test this possibility, HEK293 cells were transfected with the RyR2 wt, S2808A, or S2808D mutant cDNA.
As shown in Figure 2A, all three RyR2 variants, RyR2 wt, S2808A, and S2808D, were pulled down by GSTFKBP12.6.
Immunoblotting using the anti-S2808(PO3) antibody confirmed that the RyR2 wt was phosphorylated, but the S2808A and S2808D mutants were not.
Thus, these results demonstrate that GST-FKBP12.6 is able to interact with RyR2 regardless of the phosphorylation state of serine-2808 and with the S2808D mutant.
FKBP12.6 Is Coimmunoprecipitated With RyR2 wt and the S2808A and S2808D Mutants by an Anti-RyR Antibody RyR2 is a tetrameric structure consisting of four subunits with an identical primary sequence.
In that case, it would have been possible for GSTFKBP12.6 to interact only with the nonphosphorylated RyR2 subunits, and therefore the presence of the phosphorylated RyR2 in the GST-FKBP12.6 precipitates seen in Figure 2 would be a result of coprecipitation with the nonphosphory- Effect of Phosphorylation and Mutations at Serine-2808 on FKBP12.6-RyR2 Interaction To assess the impact of phosphorylation at serine-2808 on FKBP12.6-RyR2 interaction, we used three RyR2 variants, RyR2 wt and the S2808A and S2808D mutants.
Rapamycin-sensitive coimmunoprecipitation of FKBP12.6 with RyR2 wt and the S2808A and S2808D mutants.
A and B, Expressed RyR2(wt), S2808A, and S2808D were immunoprecipitated by the anti-RyR(34c) antibody in the presence of exogenous FKBP12.6 (250 nmol/L) (lanes 1 through 3) or in the presence of exogenous FKBP12.6 (250 nmol/L) plus rapamycin (5 mol/L) (lanes 4 through 6).
RyR2 wt and the S2808A and S2808D Mutants Are Capable of Forming Complexes With FKBP12.6 in HEK293 Cells It is clear that RyR2 containing either a phosphorylated or nonphosphorylated serine-2808 can form a complex with GST-FKBP12.6 or FKBP12.6 in vitro.
As shown in Figure 4, RyR2 wt, but not the S2808A or S2808D mutant, was phosphorylated at serine-2808.
Importantly, all three RyR2 variants, the phosphorylated RyR2 wt, the nonphosphorylated S2808A mutant, and the S2808D mutant, thought to mimic constitutive phosphorylation were able to precipitate the coexpressed FKBP12.6 (Figure 4C, lanes 4 through 6).
RyR2 wt and the S2808A and S2808D mutants are able to interact with FKBP12.6 in HEK293 cells The expressed RyR2(wt) (lane 1), S2808A (lane 2), and S2808D (lane 3) or the coexpressed FKBP12.6-RyR2(wt) (lanes 4, 7, and 8), FKBP12.6S2808A (lane 5), and FKBP12.6-S2808D (lane 6) complexes were immunoprecipitated by the anti-RyR(34c) antibody.
Complete Phosphorylation of RyR2 at Serine-2808 by Exogenous PKA Does Not Dissociate FKBP12.6 From RyR2 To additionally investigate whether phosphorylation at serine-2808 has any effect on FKBP12.6 binding to RyR2, RyR2 wt and the S2808A and S2808D mutants were coexpressed with FKBP12.6 in HEK293 cells.
Phosphorylation of RyR2 wt, S2808A, or S2808D does not dissociate FKBP12.6.
However, this key finding was questioned recently by 494 Circulation Research March 5, 2004 the results of our present study do not support the notions that FKBP12.6 cannot bind to serine-2808 phosphorylated RyR2 or the S2808D mutant and that PKA phosphorylation of RyR2 at serine-2808 dissociates FKBP12.6.8,10 The reasons for the discrepancies between our studies and those of Marx et al8 and Wehrens et al10 are unclear.
It has recently been shown that a single point mutation at the serine-2808 phosphorylation site, S2808D, abolished FKBP12.6 binding, implying that residue serine-2808 is essential for FKBP12.6 interaction.10 This is surprising, given our previous observation that a large region in RyR2 (resi- Figure 7.
Our data show that (1) FKBP12.6 is able to bind to both the serine-2808 phosphorylated and nonphosphorylated forms of RyR2; (2) FKBP12.6 can also bind to the S2808D mutant, which is thought to mimic constitutive phosphorylation; (3) complete phosphorylation at serine-2808 by exogenous PKA does not dissociate FKBP12.6 from either recombinant or native RyR2; and (4) binding of an antibody to the serine2808 phosphorylation site does not prevent FKBP12.6 binding to or dissociate FKBP12.6 from RyR2.
Taken together, Xiao et al PKA Phosphorylation and FKBP12.6-RyR2 Interaction 495 dues 1937 to 4967) encompassing the serine-2808 phosphorylation site and the previously mapped FKBP12.6 binding site (residues 2427 through 2428) is not required for FKBP12.6 binding to RyR2.17,18 To investigate the significance of the serine-2808 phosphorylation site in FKBP12.6 interaction, we have mutated serine-2808 of RyR2 to aspartate, S2808D.
In contrast to the study by Wehrens et al,10 we found that both RyR2 wt and the S2808D mutant were able to interact with FKBP12.6.
Our results clearly indicate that FKBP12.6 can bind to serine-2808 phosphorylated RyR2 and the S2808D mutant and that PKA phosphorylation of RyR2 at serine-2808 does not dissociate FKBP12.6.
S2809D
protein
substitution
true positive
P30957
On the other hand, our results are consistent with those reported recently by Stange et al,20 who showed that the S2809D mutation in rabbit RyR2 did not abolish FKBP12.6 binding.
S2808A
protein
substitution
true positive
Q92736
C, Expressed RyR2(wt) (lane 1) or S2808A (lane 2) protein was immunoprecipitated by the anti-RyR(34c) antibody.
Mutations S2808A and S2808D were introduced into the mouse RyR2 by the overlap extension method.
Analysis of FKBP12.6 interaction with RyR2 or mutants S2808A and S2808D was performed by coimmunoprecipitation assay using HEK293 cell lysate or solubilized canine heart microsome.
Anti-RyR(34c) was used for immunoprecipitation and detection of RyR2, S2808A, and S2808D.
To obtain a negative control for the antibody, we mutated serine-2808 to alanine (S2808A) to completely remove the phosphorylation site.
The RyR2 wild-type (wt) and S2808A mutant were expressed in HEK293 cells, and the expressed RyR2 wt and mutant proteins were immunoprecipitated by an anti-RyR antibody, anti-RyR(34c).
The anti-S2808(deP) antibody was generated against a nonphosphorylated serine2809 peptide of the rabbit RyR2 and has been characterized to be specific to the nonphosphorylated form of RyR2.11 As seen in Figure 1C, the RyR2 wt and S2808A mutant were expressed in HEK293 cells at an equivalent level (Figure 1Ca).
Neither the anti-S2808(PO3) nor the anti-S2808(deP) antibody interacted with the S2808A mutant (Figures 1Cb and 1Cc, lane 2), indicating that the affinity-purified antiS2808(PO3) antibody and the anti-S2808(deP) antibody are specific to the S2808-peptide in the context of the intact RyR2 protein.
Precipitation of the RyR2(wt), S2808A, and S2808D by GST-FKBP12.6.
The expressed RyR2(wt) (lane 1), S2808A (lane 2), and S2808D (lane 3) proteins were precipitated from cell lysates by GST-FKBP12.6 or by GST alone.
On the other hand, the S2808D mutant, which is thought to mimic phosphorylation, may correspond to a constitutively phosphorylated state of serine-2808.10 If the phosphorylation state of serine-2808 dictates the interaction between FKBP12.6 and RyR2, RyR2 wt and the S2808A and S2808D mutants would be expected to differ markedly in their ability to interact with FKBP12.6.
To test this possibility, HEK293 cells were transfected with the RyR2 wt, S2808A, or S2808D mutant cDNA.
As shown in Figure 2A, all three RyR2 variants, RyR2 wt, S2808A, and S2808D, were pulled down by GSTFKBP12.6.
Immunoblotting using the anti-S2808(PO3) antibody confirmed that the RyR2 wt was phosphorylated, but the S2808A and S2808D mutants were not.
FKBP12.6 Is Coimmunoprecipitated With RyR2 wt and the S2808A and S2808D Mutants by an Anti-RyR Antibody RyR2 is a tetrameric structure consisting of four subunits with an identical primary sequence.
In that case, it would have been possible for GSTFKBP12.6 to interact only with the nonphosphorylated RyR2 subunits, and therefore the presence of the phosphorylated RyR2 in the GST-FKBP12.6 precipitates seen in Figure 2 would be a result of coprecipitation with the nonphosphory- Effect of Phosphorylation and Mutations at Serine-2808 on FKBP12.6-RyR2 Interaction To assess the impact of phosphorylation at serine-2808 on FKBP12.6-RyR2 interaction, we used three RyR2 variants, RyR2 wt and the S2808A and S2808D mutants.
Because mutant S2808A cannot be phosphorylated at serine2808, it represents a completely nonphosphorylated state of serine-2808, whereas the RyR2 wt when expressed in HEK293 490 Circulation Research March 5, 2004 Figure 3.
Rapamycin-sensitive coimmunoprecipitation of FKBP12.6 with RyR2 wt and the S2808A and S2808D mutants.
A and B, Expressed RyR2(wt), S2808A, and S2808D were immunoprecipitated by the anti-RyR(34c) antibody in the presence of exogenous FKBP12.6 (250 nmol/L) (lanes 1 through 3) or in the presence of exogenous FKBP12.6 (250 nmol/L) plus rapamycin (5 mol/L) (lanes 4 through 6).
We reasoned that if FKBP12.6 interacts only with the serine-2808 nonphosphorylated RyR2 subunits, the amount of FKBP12.6 associated with the partially phosphorylated RyR2 wt would be relatively less than that associated with the completely nonphosphorylated S2808A mutant.
RyR2 wt and the S2808A and S2808D Mutants Are Capable of Forming Complexes With FKBP12.6 in HEK293 Cells It is clear that RyR2 containing either a phosphorylated or nonphosphorylated serine-2808 can form a complex with GST-FKBP12.6 or FKBP12.6 in vitro.
As shown in Figure 4, RyR2 wt, but not the S2808A or S2808D mutant, was phosphorylated at serine-2808.
Importantly, all three RyR2 variants, the phosphorylated RyR2 wt, the nonphosphorylated S2808A mutant, and the S2808D mutant, thought to mimic constitutive phosphorylation were able to precipitate the coexpressed FKBP12.6 (Figure 4C, lanes 4 through 6).
The affinity-purified antibody interacted with both the phosphorylated and nonphosphorylated forms of serine-2808 peptide (Figures 6Aa and 6Ab) but did not recognize the S2808A mutant (Figure 6Ac), indicating that the antibody is specific in the intact RyR2 protein.
RyR2 wt and the S2808A and S2808D mutants are able to interact with FKBP12.6 in HEK293 cells The expressed RyR2(wt) (lane 1), S2808A (lane 2), and S2808D (lane 3) or the coexpressed FKBP12.6-RyR2(wt) (lanes 4, 7, and 8), FKBP12.6S2808A (lane 5), and FKBP12.6-S2808D (lane 6) complexes were immunoprecipitated by the anti-RyR(34c) antibody.
Complete Phosphorylation of RyR2 at Serine-2808 by Exogenous PKA Does Not Dissociate FKBP12.6 From RyR2 To additionally investigate whether phosphorylation at serine-2808 has any effect on FKBP12.6 binding to RyR2, RyR2 wt and the S2808A and S2808D mutants were coexpressed with FKBP12.6 in HEK293 cells.
Phosphorylation of RyR2 wt, S2808A, or S2808D does not dissociate FKBP12.6.
The immunoreactivity of the antiS2808-peptide antibody with the S2808(PO3)-peptide (Aa), the S2808peptide (Ab), the RyR2 wt, and the S2808A mutant (Ac) is shown in A.
Using these antibodies, we verified that the S2808A RyR2 mutant was completely nonphosphorylated, whereas RyR2 treated with exogenous PKA was completely phosphorylated at serine-2808.
9482942
full text
A438K
protein
substitution
P15390
true positive
Three of these 12 mut ants did not ex press suf ficient currents (L 432K, A438K, and V440K residues).
N434K
protein
substitution
P15390
true positive
We show that a single asparagine 3 lysine substitution of the rat muscle Na channel -subunit, 1-N434K, renders the channel completely insensitive to 5 M BTX when expressed in mammalian cells.
To avoid unwanted mismatch mut ations, we selected t wo independent clones for 1-N434K and for 1-N434A.
R ESULTS AND DISCUSSION Mut ation 1-N434K Yields a BTX-Resist ant Phenot ype w ith Nor mal Na Cur rent K inetics.
1B shows the current family rec orded f rom a cell transfected w ith 1-N434K mut ant Na channels.
The threshold for activation was also near 50 to 40 mV and the 0 value of the fast-decaying phase at 50 mV measured 0.23 .01 ms (mean SEM) for both w ild t ype (n 8) and N434K mut ant (n 10).
By c omparison, even af ter 1,800 repetitive pulses BTX did not elicit sign ificant changes in the 1-N434K current in 20 cells tested (Fig.
The N434K mut ant channel was thus c ompletely resist ant to BTX at 5 M.
The parameters for the activation and the steady-st ate inactivation (h ) of N434K mut ant were strik ingly c omparable to those of the w ild t ype (Table 1), except a small 3 mV shif t 0.05).
1), demonstrated that BTX-resist ant 1-N434K channels were functionally nor mal.
To test whether 1-N434K channels are sensitive to veratridine, we applied 200 M of this alk aloid f rom the external side under equal internal external Na ion c oncentrations.
2 A and B show that veratridine inhibits the peak 1 and 1-N434K currents equally by about 2535% (28.7 2.0%, n 8, and Diag ram 2 Phar mac olog y: Wang and Wang P roc.
Na current families were rec orded in Hek293t cells ex pressing either 1-wild-type ( A ) or 1-N434K channels ( B ) under reversed Na g radient c onditions.
With 5 M BTX in the pipette, repetitive pulses (Lower) were applied at 2 Hz and current traces of 1-wild-type ( C ) or 1-N434K channels ( D ) were superimposed.
In c ontrast, the N434K mut ant lacked the non inactivating current and had little veratridine-induced t ail current (Fig.
This phenot ype suggests that veratridine-modified N434K mut ant Na channels may have an even smaller c onduct ance (i.e., FIG.
The inhibition of peak 1-wild-type ( A ) and 1-N434K ( B ) Na currents by external 200 M veratridine was measured by a test pulse ( 50 mV for 6 ms) under equal internal external Na ion c oncentrations.
Repetitive pulses (shown below) elicited only a small use-dependent inhibition of peak 1-wild-type ( C ) and 1-N434K ( D ) Na currents.
With superfusion of 200 M veratridine for 5 min, repetitive pulses elicited sign ificant use-dependent inhibition of peak 1-wild-type ( E ) and 1-N434K ( F ) Na currents.
Even w ith an increase in the amplifier gain by 20- to 40-fold the rec ords did not show any non inactivating veratridine-induced N434K channel activ it y during the pulse.
Without veratridine, repetitive pulses at 5 Hz elicited little use-dependent inhibition of w ild-t ype and 1-N434K Na currents (Fig.
In the presence of veratridine, repetitive pulses elicited sign ificant use-dependent inhibition of both w ild-t ype and 1-N434K peak currents (Fig.
The persistent t ail current at 1 w ild-t ype and 1-N434K 11) 11) 10) 10) 35.0 8.7 73.3 6.5 2.5 mV ( n 0.8 mV ( n 1.1 mV ( n 0.3 mV ( n 9) 9) 10)* 10) Table 1.
Volt age dependence of activation and steady-st ate inactivation in 1-N434K channels Parameter Activation Steady-st ate inactivation ( h ) 1 Wild t ype E 0.5 k a factor h 0.5 k h factor 39.2 7.8 76.6 7.2 2.3 mV ( n 1.0 mV ( n 0.9 mV ( n 0.3 mV ( n Cells were dialyzed by internal solution for 30 min, and the activation parameters were deter mined by I V cur ves in equal internal external Na ion c oncentrations.
In our assay, veratridine therefore is equally ef fective in inhibiting the peak 1-N434K and w ild-t ype Na currents, despite the fact that veratridine-modified 1-N434K channels may carr y ver y little current (Fig.
To ex plain this phenot ype we suggest that the N434K mut ation does not abolish veratridine binding but does appear to af fect the channel c onduct ance or the gating behav ior on veratridine binding.
Because BTX and veratridine are c ompetitive and mutually exclusive in binding and because BTX binding w ith native Na channels is practically irreversible under volt age-clamp c onditions (6), it is possible to per for m protection ex periments w ith these t wo tox ins in w ild-t ype and in 1-N434K mut ant channels.
To deter mine whether BTX binding is tr uly abolished in the 1-N434K channel, we included 5 M BTX in the pipette and applied 1,200 repetitive pulses.
28.7 2.0% w ithout BTX; P 0.05), but the inhibition of BTX-pretreated 1-N434K current persists (32.5 1.9%, n 5, vs.
Likew ise, the use-dependent inhibition of the 1-N434K peak current (55% inhibition) by veratridine remains w ith BTX present (Fig.
c This result supports the prev ious c onclusion that in Na hannels BTX and veratridine are mutually exclusive in binding (4) and implies that BTX does not bind to 1-N434K mut ant channels.
Traces of BTX-pretreated 1-wild-type ( A ) and 1-N434K ( B ) Na currents under a reversed Na g radient are superimposed before and af ter external 200 M veratridine application.
With super fusion of 200 M veratridine, repetitive pulses were reapplied to elicit BTX-treated 1-wild-type ( C ) and 1-N434K ( D ) currents.
Additionally, we obser ved that an equivalent mut ation to 1-N434K in human heart Na channels (22), hH1-N406K, also renders these channels c ompletely resist ant to BTX (tested in n ine cells), thereby demonstrating that a single N3 K mut ation in segment I-S6 produces the same BTX-resist ant phenot ype regardless of Na channel isofor m.
This hH1-N406K channel also remains sensitive to 200 M veratridine in a manner similar to 1-N434K channel.
Because alan ine at N434A is a nonpolar residue, it may not abolish BTX binding as charged lysine at N434K does.
Why then does the 1-N434K-mutation produce little changes in Na channel gating (Fig.
N434A
protein
substitution
P15390
true positive
Another substitution, 1-N434A, yields a partial BTXsensitive mutant.
Unlike wild-type currents, the BTX-modified 1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional.
To avoid unwanted mismatch mut ations, we selected t wo independent clones for 1-N434K and for 1-N434A.
Prev iously, we reported that a mut ation in 1-N434A enhances slow inactivation and ac celerates fast inactivation (13).
Because alan ine at N434A is a nonpolar residue, it may not abolish BTX binding as charged lysine at N434K does.
4 shows that BTX still binds w ith 1-N434A channels but its ef fects dif fer c onsiderably f rom those in w ild-t ype channels.
4 A), BTX-modified N434A currents c ontinued to decline w ith a multiphasic k inetic (Fig.
It is possible that this decaying phase of BTX-modified 1-N434A currents is caused by the fast and slow inactivation of BTX-modified 1-N434A channels.
To test this possibilit y we investigated the slow inactivation of BTX-modified 1-N434A channels during a prolonged depolarization by a t wo-pulse protoc ol as shown in Fig.
A 50-ms gap of 130 mV was inserted to allow the rec over y of BTX-modified 1-N434A channels f rom the obser ved fast inactivation shown in Fig.
In c ontrast, more than 90% of BTX-modified N434A mut ant currents are inactivated under the same pulse protoc ol.
Ac c ordingly, the remnant of this slow inactivation process is likely to be the cause of the slow decaying phase of the BTX-modified 1-N434A currents shown in Fig.
Superimposed traces of 1-wild-type ( A ) and 1-N434A (B and C) Na current families at various volt ages were rec orded under reversed Na gradient c onditions.
Frequent rests during repetitive pulses were allowed in 1-N434A for rec over y f rom slow inactivation (13).
The BTX-modified 1-N434A currents w ith multiple decaying phases shown w ith t wo dif ferent time scales are ev ident (B and C; f1 12.8 ms, f2 86.4 ms, and s 1.56 s at 30 mV).
Note that BTX-modified w ild-t ype and 1-N434A Na channels were activated at potentials 30 40 mV more negative than the unmodified w ild-t ype channels.
( D ) The time c ourses of slow inactivation in BTX-modified 1 and 1-N434A channels were measured by a c onventional t wo-pulse protoc ol (Inset).
Note that 90% of BTXmodified N434A currents were inactivated whereas little inactivation oc curred in BTX-modified 1 w ild-t ype currents.
For c omparison, the time c ourse of slow inactivation of 1-N434A channels w ithout BTX present also is shown.
A441K
protein
substitution
true positive
P15390
Na current traces f rom mut ants of 1-F430K ( A ) , 1A441K ( B ) , 1-I433K ( C ) , and 1-L437K ( D ) were rec orded during repetitive pulses at 2 Hz (protoc ol shown below).
Sign ificant use-dependent inhibition of peak currents f rom 1-A441K and 1-I433K mut ants oc curred during repetitive pulses.
A lthough 1-F430K and 1-A441K residues are located at the same face, they apparently do not exhibit the BTX-resist ant phenot ype (Fig.
L437K
protein
substitution
true positive
P15390
Mutants of two adjacent residues, 1-I433K and 1-L437K, also were found to exhibit the identical BTX-resistant phenotype.
Na current traces f rom mut ants of 1-F430K ( A ) , 1A441K ( B ) , 1-I433K ( C ) , and 1-L437K ( D ) were rec orded during repetitive pulses at 2 Hz (protoc ol shown below).
Surprisingly, we found that c only t wo mut ants, 1-I433K and 1-L437K, ex pressed Na hannels that were c ompletely resist ant to 5 M BTX (Fig.
I433K
protein
substitution
P15390
true positive
Mutants of two adjacent residues, 1-I433K and 1-L437K, also were found to exhibit the identical BTX-resistant phenotype.
Na current traces f rom mut ants of 1-F430K ( A ) , 1A441K ( B ) , 1-I433K ( C ) , and 1-L437K ( D ) were rec orded during repetitive pulses at 2 Hz (protoc ol shown below).
Sign ificant use-dependent inhibition of peak currents f rom 1-A441K and 1-I433K mut ants oc curred during repetitive pulses.
Surprisingly, we found that c only t wo mut ants, 1-I433K and 1-L437K, ex pressed Na hannels that were c ompletely resist ant to 5 M BTX (Fig.
Within 12 adjacent mut ants tested only t wo other lysine mut ants (I433K and L 437K) exhibit BTX-resist ant phenot ype, suggesting that these three residues are w ithin the BTX binding domain.
N406K
protein
substitution
true positive
Q14524
Additionally, we obser ved that an equivalent mut ation to 1-N434K in human heart Na channels (22), hH1-N406K, also renders these channels c ompletely resist ant to BTX (tested in n ine cells), thereby demonstrating that a single N3 K mut ation in segment I-S6 produces the same BTX-resist ant phenot ype regardless of Na channel isofor m.
This hH1-N406K channel also remains sensitive to 200 M veratridine in a manner similar to 1-N434K channel.
F430K
protein
substitution
P15390
true positive
Na current traces f rom mut ants of 1-F430K ( A ) , 1A441K ( B ) , 1-I433K ( C ) , and 1-L437K ( D ) were rec orded during repetitive pulses at 2 Hz (protoc ol shown below).
A lthough 1-F430K and 1-A441K residues are located at the same face, they apparently do not exhibit the BTX-resist ant phenot ype (Fig.
V440K
protein
substitution
true positive
P15390
Three of these 12 mut ants did not ex press suf ficient currents (L 432K, A438K, and V440K residues).
N434R
protein
substitution
P15390
true positive
idea, an identical BTX-resist ant, veratridine-sensitive phenot ype was found for the 1-N434R (argin ine) mut ant (tested in seven cells).
L432K
protein
substitution
true positive
P15390
Three of these 12 mut ants did not ex press suf ficient currents (L 432K, A438K, and V440K residues).
10051517
full text
10097041
full text
I403M
protein
substitution
true positive
P21817
Five mut ations leading to both MH and CCD susceptibilit y are R163C, I403M, Y522S, R2163H, and R2436H.
Y522S
protein
substitution
true positive
P21817
Five mut ations leading to both MH and CCD susceptibilit y are R163C, I403M, Y522S, R2163H, and R2436H.
D4900A
protein
substitution
true positive
P21817
2 A is that other mut ations (D4900A, D4900R, R4914E, and D4918A in Fig.
I4898T
protein
substitution
true positive
P21817
(1) report a new mut ation I4898T f rom a large Mex ican pedig ree that is associated w ith CCD.
Ex pression of the I4898T mut ant in HEK cells yielded g r e a t l y r e d u c e d [3H ] r y a n o d i n e b i n d i n g l e v e l s .
Coex pression of w ild-t ype RyR1 and the I4898T RyR1 mut ant in HEK cells decreased the threshold of Ca2 required to in itiate open ing of w ild-t ype RyR1 and resulted in a reduced release of Ca2 f rom internal stores, implying that the central defect for MH and CCD mut ations in RyR1 is a leaky channel.
Molecular Predictions Based on the I4898T RyR1 Mut ation The N-ter minal 4,000-aa residues of RyR1 for m a large, loosely packed c y tosolic foot domain (12).
A lternatively, the I4898T mut ation may alter regulation of the RyR1 channel by luminal Ca2 .
A regulator y luminal Ca2 binding site c ould possibly be disr upted by the I4898T mut ation.
A third, and favored, ex planation for the ef fects of the I4898T mut ation is that this residue c onstitutes part of the ion c onducting pore of c yR1.
though it is possible that the I4898T mut ation may disr upt the regulation of RyR1 by associated luminal proteins in muscle, A B FIG.
It therefore is highly likely that the I4898T mut ation has w ide-ranging c onsequences for the str ucture of the pore as well as the ion selectiv it y of the channel.
The import ance of the I4898T RyR1 mut ation is borne out by the finding that high-af fin it y r yanodine binding in isolated vesicles and phar mac ologically induced calcium release in a cell-based assay are absent for this mut ant (1).
(1) demonstrated that the I4898T mut ation does alter Ca2 dependence of r yanodine binding, which is believed to be an indicator of channel activation.
In c onclusion, the I4898T mut ation in the RyR1 described by Lynch et al .
R163C
protein
substitution
true positive
P21817
Five mut ations leading to both MH and CCD susceptibilit y are R163C, I403M, Y522S, R2163H, and R2436H.
R4914E
protein
substitution
true positive
P21817
2 A is that other mut ations (D4900A, D4900R, R4914E, and D4918A in Fig.
R2436H
protein
substitution
true negative
Five mut ations leading to both MH and CCD susceptibilit y are R163C, I403M, Y522S, R2163H, and R2436H.
D4918A
protein
substitution
true positive
P21817
2 A is that other mut ations (D4900A, D4900R, R4914E, and D4918A in Fig.
R2163H
protein
substitution
true positive
P21817
Five mut ations leading to both MH and CCD susceptibilit y are R163C, I403M, Y522S, R2163H, and R2436H.
D4900R
protein
substitution
true positive
P21817
2 A is that other mut ations (D4900A, D4900R, R4914E, and D4918A in Fig.
10388752
full text
M1425K
protein
substitution
P04775
true positive
We also verified that the mutations E387Q and M1425K, involving a net increase of one positive charge in the homologous positions of repeats I and III, have such a low TTX sensitivity as to make their further study impractical.
E387Q
protein
substitution
P04775
true positive
We also verified that the mutations E387Q and M1425K, involving a net increase of one positive charge in the homologous positions of repeats I and III, have such a low TTX sensitivity as to make their further study impractical.
E945Q
protein
substitution
P04775
true positive
Biophysical Journal Volume 77 July 1999 229 240 229 Tonic and Phasic Tetrodotoxin Block of Sodium Channels with Point Mutations in the Outer Pore Region Anna Boccaccio, Oscar Moran, Keiji Imoto,# and Franco Conti Istituto di Cibernetica e Biofisica, CNR, I-16149 Genova, Italy #National Institute for Physiological Sciences, Okazaki 444, Japan ABSTRACT Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA -subunit of sodium ) channels.
Except for mutant E945Q, all phenotypes have roughly the same value of ) kon 2 M 1 s 1 and owe their large differences in IC(t0 to different koff values.
However, a 60-fold reduction in kon is the 5 main determinant of the low TTX sensitivity of mutant E945Q.
We report here this type of study for the channels expressed in oocytes by four mutants of the rBIIA -subunit: W386Y, E945Q, D1426K, and D1717Q.
Two of the mutations (E945Q of repeat II and D1717Q of repeat IV) are at positions assigned by Terlau et al.
An interesting outcome of our analysis is that the charge neutralizations in the homologous residues E945 and D1717 cause a similar large reduction of TTX sensitivity by having opposite effects on the kinetics of TTX binding: the rate of TTX association to mutant E945Q is much lower, whereas the off-binding of TTX from D1717Q is much faster, than for WT channels.
Mutation W386Y in the first repeat is fully conservative; mutations E945Q and D1717Q, respectively, in the second and fourth repeat, involve the neutralization of a negative charge; mutation D1426K in the third repeat involves a positive double charge increase.
The four mutations (W386Y, E945Q, D1426K, D1717Q) of the pore region that we studied in this work are illustrated in Fig.
The correction was barely significant for the currents expressed by WT, W386Y, D1426K, and E945Q, whose maximum use-dependent block could be measured for stimulation frequencies lower than 2 Hz; it was substantial, however, in the experiments with mutant D1717Q, which has fast TTX-binding kinetics and shows large UD effects only for stimulation frequencies above 10 Hz.
2 the resting periods were 20 s for mutant D1717Q, 1 min for W386Y and E945Q, 3 min for WT, and 6 min for D1426K.
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
Use-dependent block of D1426K, W386Y, and E945Q Like that of WT channels, the block by TTX of the sodium currents mediated by three of the mutants studied in this work can be easily shown to have use-dependent properties.
3 an oocyte expressing WT, W386Y, D1426K, or E945Q was exposed to a TTX con) centration close to the tonic IC(t0 of each phenotype, and the 5 train stimulation was started after a suitable resting period at a holding potential.
Peak currents elicited by the various pulses of the train are normalized to those measured with TABLE 1 Summary of the parameters characterizing the affinity and the kinetics of TTX binding to resting and stimulated sodium channel mutants ) IC(t0 (nM) 5 ) IC(s0 (nM) 5 0 (s) 53 7.2 12.0 225 103 16 0.3 0.8 7 6 10 Bi 0.66 0.69 0.67 0.56 0.60 k(0)* (s) off 0.019 0.14 0.083 0.0044 9.71 kon* ( M s) 2.1 1.8 0.038 1.9 2.1 WT* W386Y E945Q D1426K D1717Q 26 247 6.3 3.9 11.3 4 19 0.6 0.6 0.7 103 103 8.9 77 2.2 1.7 4.6 1.2 5 0.2 0.3 0.3 103 103 3 Data are means standard deviation.
TTX-Block of Sodium Outer-Pore Mutants 233 FIGURE 3 Use dependence of TTX block of mutants W386Y, E945Q, and D1426K, compared to that of the WT channel, as revealed by the response to repeated stimulations with the same short pulse at 0.6-s intervals, starting from resting conditions of 50% tonic block.
The values of the parameters U0, U , and that best fit the data are 0.5, 0.28, and 16.3 s for WT; 0.48, 0.31, and 2.2 s for W386Y; 0.63, 0.35, and 5.1 s for E945Q; and 0.6, 0.34, and 61 s for D1426K.
4 shows representative experiments illustrating this property for WT, W386Y, and E945Q.
(B) E945Q and 10 M-TTX: f 1.3 s, s 13 s.
Measurements of single-pulse relaxations are generally less accurate than those of cumulative extra block, because the effects are much smaller and the experiments are too long (each data point requires a resting period of 1 min for E945Q and 3 min for WT; our estimate that D1426K recovers the tonic block conditions only after 6 min discouraged us from performing double-pulse measurements in this mutant).
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
For mutants W386Y, D1426K, and E945Q, experiments of the type illustrated in Fig.
With the notice5 off able exception of E945Q, this appears to be the case for all other mutants for which we estimate kon 2 M 1 s 1, as for the WT (Table 1).
The case of E945Q, which has a low TTX sensitivity comparable to that of D1717Q (see Table ) 1), appears quite anomalous.
The increase in both IC(t0 and 5 (s) IC50 by a factor of 250 in mutant E945Q is due to the combination of a modest approximately fourfold increase in k(0) and a much larger 60-fold decrease in kon.
W386Y
protein
substitution
P04775
true positive
Biophysical Journal Volume 77 July 1999 229 240 229 Tonic and Phasic Tetrodotoxin Block of Sodium Channels with Point Mutations in the Outer Pore Region Anna Boccaccio, Oscar Moran, Keiji Imoto,# and Franco Conti Istituto di Cibernetica e Biofisica, CNR, I-16149 Genova, Italy #National Institute for Physiological Sciences, Okazaki 444, Japan ABSTRACT Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA -subunit of sodium ) channels.
We report here this type of study for the channels expressed in oocytes by four mutants of the rBIIA -subunit: W386Y, E945Q, D1426K, and D1717Q.
(1991) to the outer ring of strongly TTX-sensitive residues, whereas W386Y and D1426K are one position below and one above the residues contributed to this ring by repeats I and III.
Mutation W386Y in the first repeat is fully conservative; mutations E945Q and D1717Q, respectively, in the second and fourth repeat, involve the neutralization of a negative charge; mutation D1426K in the third repeat involves a positive double charge increase.
The four mutations (W386Y, E945Q, D1426K, D1717Q) of the pore region that we studied in this work are illustrated in Fig.
Several experiments with the most TTX-sensitive phenotypes (WT, W386Y, and D1426K) were ended by perfusion with high concentrations of TTX that completely blocked the sodium channels, and the remaining currents could be used for a more accurate leakage subtraction.
The correction was barely significant for the currents expressed by WT, W386Y, D1426K, and E945Q, whose maximum use-dependent block could be measured for stimulation frequencies lower than 2 Hz; it was substantial, however, in the experiments with mutant D1717Q, which has fast TTX-binding kinetics and shows large UD effects only for stimulation frequencies above 10 Hz.
2 the resting periods were 20 s for mutant D1717Q, 1 min for W386Y and E945Q, 3 min for WT, and 6 min for D1426K.
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
Use-dependent block of D1426K, W386Y, and E945Q Like that of WT channels, the block by TTX of the sodium currents mediated by three of the mutants studied in this work can be easily shown to have use-dependent properties.
3 an oocyte expressing WT, W386Y, D1426K, or E945Q was exposed to a TTX con) centration close to the tonic IC(t0 of each phenotype, and the 5 train stimulation was started after a suitable resting period at a holding potential.
Peak currents elicited by the various pulses of the train are normalized to those measured with TABLE 1 Summary of the parameters characterizing the affinity and the kinetics of TTX binding to resting and stimulated sodium channel mutants ) IC(t0 (nM) 5 ) IC(s0 (nM) 5 0 (s) 53 7.2 12.0 225 103 16 0.3 0.8 7 6 10 Bi 0.66 0.69 0.67 0.56 0.60 k(0)* (s) off 0.019 0.14 0.083 0.0044 9.71 kon* ( M s) 2.1 1.8 0.038 1.9 2.1 WT* W386Y E945Q D1426K D1717Q 26 247 6.3 3.9 11.3 4 19 0.6 0.6 0.7 103 103 8.9 77 2.2 1.7 4.6 1.2 5 0.2 0.3 0.3 103 103 3 Data are means standard deviation.
TTX-Block of Sodium Outer-Pore Mutants 233 FIGURE 3 Use dependence of TTX block of mutants W386Y, E945Q, and D1426K, compared to that of the WT channel, as revealed by the response to repeated stimulations with the same short pulse at 0.6-s intervals, starting from resting conditions of 50% tonic block.
The values of the parameters U0, U , and that best fit the data are 0.5, 0.28, and 16.3 s for WT; 0.48, 0.31, and 2.2 s for W386Y; 0.63, 0.35, and 5.1 s for E945Q; and 0.6, 0.34, and 61 s for D1426K.
3 range from 2.2 s for mutant W386Y to 61 s for mutant D1426K.
4 shows representative experiments illustrating this property for WT, W386Y, and E945Q.
(C) W386Y and 60 nM-TTX: f 0.7 s, s 7.9 s.
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
For mutants W386Y, D1426K, and E945Q, experiments of the type illustrated in Fig.
D1426K
protein
substitution
P04775
true positive
Biophysical Journal Volume 77 July 1999 229 240 229 Tonic and Phasic Tetrodotoxin Block of Sodium Channels with Point Mutations in the Outer Pore Region Anna Boccaccio, Oscar Moran, Keiji Imoto,# and Franco Conti Istituto di Cibernetica e Biofisica, CNR, I-16149 Genova, Italy #National Institute for Physiological Sciences, Okazaki 444, Japan ABSTRACT Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA -subunit of sodium ) channels.
The various phenotypes are tonically half-blocked at TTX concentrations, IC(t0, that span a range of more than three 5 orders of magnitude, from 4 nM in mutant D1426K to 11 M in mutant D1717Q.
When stimulated with repetitive depolarizing pulses at saturating frequencies, all channels showed a monoexponential increase in their TTX-binding affinity with time ) constants that span an equally wide range of values ([TTX] IC(t0, from 60 s for D1426K to 30 ms for D1717Q) and are 5 (t) in most phenotypes roughly inversely proportional to IC50.
We report here this type of study for the channels expressed in oocytes by four mutants of the rBIIA -subunit: W386Y, E945Q, D1426K, and D1717Q.
(1991) to the outer ring of strongly TTX-sensitive residues, whereas W386Y and D1426K are one position below and one above the residues contributed to this ring by repeats I and III.
Mutation W386Y in the first repeat is fully conservative; mutations E945Q and D1717Q, respectively, in the second and fourth repeat, involve the neutralization of a negative charge; mutation D1426K in the third repeat involves a positive double charge increase.
The four mutations (W386Y, E945Q, D1426K, D1717Q) of the pore region that we studied in this work are illustrated in Fig.
Several experiments with the most TTX-sensitive phenotypes (WT, W386Y, and D1426K) were ended by perfusion with high concentrations of TTX that completely blocked the sodium channels, and the remaining currents could be used for a more accurate leakage subtraction.
The correction was barely significant for the currents expressed by WT, W386Y, D1426K, and E945Q, whose maximum use-dependent block could be measured for stimulation frequencies lower than 2 Hz; it was substantial, however, in the experiments with mutant D1717Q, which has fast TTX-binding kinetics and shows large UD effects only for stimulation frequencies above 10 Hz.
2 the resting periods were 20 s for mutant D1717Q, 1 min for W386Y and E945Q, 3 min for WT, and 6 min for D1426K.
Notice that the [T] values used in the various experiments vary by more than three orders of magnitude, from 4 nM in the experiment with D1426K to 10 M in the case of mutant D1717Q.
1), with IC(t0 values 5 ranging from 4 nM for D1426K to 11 M for D1717Q.
The slow kinetics of TTX binding to WT and D1426K channels also allowed for these phenotypes estimates of 0 from the time constant of the change of unblocked currents during wash-in and wash-out experiments.
Except for the D1426K data, reporting single measurements, the various symbols represent mean values ( SD) from at least three different measurements at a given [T].
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
1, yielding the estimates given in the second column of Table 1, which range from 4 nM for D1426K to 11 M for D1717Q .
Use-dependent block of D1426K, W386Y, and E945Q Like that of WT channels, the block by TTX of the sodium currents mediated by three of the mutants studied in this work can be easily shown to have use-dependent properties.
3 an oocyte expressing WT, W386Y, D1426K, or E945Q was exposed to a TTX con) centration close to the tonic IC(t0 of each phenotype, and the 5 train stimulation was started after a suitable resting period at a holding potential.
Peak currents elicited by the various pulses of the train are normalized to those measured with TABLE 1 Summary of the parameters characterizing the affinity and the kinetics of TTX binding to resting and stimulated sodium channel mutants ) IC(t0 (nM) 5 ) IC(s0 (nM) 5 0 (s) 53 7.2 12.0 225 103 16 0.3 0.8 7 6 10 Bi 0.66 0.69 0.67 0.56 0.60 k(0)* (s) off 0.019 0.14 0.083 0.0044 9.71 kon* ( M s) 2.1 1.8 0.038 1.9 2.1 WT* W386Y E945Q D1426K D1717Q 26 247 6.3 3.9 11.3 4 19 0.6 0.6 0.7 103 103 8.9 77 2.2 1.7 4.6 1.2 5 0.2 0.3 0.3 103 103 3 Data are means standard deviation.
TTX-Block of Sodium Outer-Pore Mutants 233 FIGURE 3 Use dependence of TTX block of mutants W386Y, E945Q, and D1426K, compared to that of the WT channel, as revealed by the response to repeated stimulations with the same short pulse at 0.6-s intervals, starting from resting conditions of 50% tonic block.
The values of the parameters U0, U , and that best fit the data are 0.5, 0.28, and 16.3 s for WT; 0.48, 0.31, and 2.2 s for W386Y; 0.63, 0.35, and 5.1 s for E945Q; and 0.6, 0.34, and 61 s for D1426K.
3 range from 2.2 s for mutant W386Y to 61 s for mutant D1426K.
Measurements of single-pulse relaxations are generally less accurate than those of cumulative extra block, because the effects are much smaller and the experiments are too long (each data point requires a resting period of 1 min for E945Q and 3 min for WT; our estimate that D1426K recovers the tonic block conditions only after 6 min discouraged us from performing double-pulse measurements in this mutant).
For D1426K we have also plotted in Fig.
6 A shows the time course of a wash-in/wash-out experiment on an oocyte expressing D1426K, the most TTX-sensitive phenotype of this study that is also characterized by the slowest TTX binding kinetics.
3 and 4, these data yield for ) D1426K the estimates IC(s0 2 nM and 0 220 s.
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
3, using the IC(s0 values given in the third column of Table 1, 5 ranging from 1.7 nM for D1426K to 4.6 M for D1717Q.
(B) Similar comparison for 5 the [T] dependence of the time constant, , of the stimulated decay of toxin-free probability, normalized for each phenotype, to the respective estimated upper limit, 0, given in the fourth column of Table 1 and ranging from 103 ms for D1717Q to 225 s for D1426K.
For mutant D1426K the data include as open symbols the time constants of binding relaxations measured in wash-in/wash-out experiments.
For mutants W386Y, D1426K, and E945Q, experiments of the type illustrated in Fig.
5 A versus the logarithm of [T]; they were 236 Biophysical Journal Volume 77 July 1999 FIGURE 6 Time course of the normalized peak response of mutants D1426K (A) and D1717Q (B) to a fixed pulse stimulation during wash-in/ wash-out experiments.
In four different experiments of this type, the estimated wash-out time constant for TTX unblock of D1426K channels ranged between 206 and 247 s, in good agreement with the value of 225 s (Table 1), which fits the overall data of UD and wash-in kinetics according to Eq.
The kinetics of TTX binding to WT channels is faster than for D1426K; the time constants estimated from use) dependent block relaxations at [T] IC(t0 are smaller than 5 20 s (Conti et al., 1996).
For the slow mutant D1426K, consistent values of the time constants and direct estimates of 0 were also obtained from wash-in/ wash-out experiments.
E758Q
protein
substitution
true negative
Also in the case of -CTX, the mutation E758Q causes a modest twofold increase in koff and an almost 100-fold decrease in kon (Dudley et al., 1995).
D1717Q
protein
substitution
P04775
true positive
Biophysical Journal Volume 77 July 1999 229 240 229 Tonic and Phasic Tetrodotoxin Block of Sodium Channels with Point Mutations in the Outer Pore Region Anna Boccaccio, Oscar Moran, Keiji Imoto,# and Franco Conti Istituto di Cibernetica e Biofisica, CNR, I-16149 Genova, Italy #National Institute for Physiological Sciences, Okazaki 444, Japan ABSTRACT Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA -subunit of sodium ) channels.
The various phenotypes are tonically half-blocked at TTX concentrations, IC(t0, that span a range of more than three 5 orders of magnitude, from 4 nM in mutant D1426K to 11 M in mutant D1717Q.
When stimulated with repetitive depolarizing pulses at saturating frequencies, all channels showed a monoexponential increase in their TTX-binding affinity with time ) constants that span an equally wide range of values ([TTX] IC(t0, from 60 s for D1426K to 30 ms for D1717Q) and are 5 (t) in most phenotypes roughly inversely proportional to IC50.
We report here this type of study for the channels expressed in oocytes by four mutants of the rBIIA -subunit: W386Y, E945Q, D1426K, and D1717Q.
Two of the mutations (E945Q of repeat II and D1717Q of repeat IV) are at positions assigned by Terlau et al.
An interesting outcome of our analysis is that the charge neutralizations in the homologous residues E945 and D1717 cause a similar large reduction of TTX sensitivity by having opposite effects on the kinetics of TTX binding: the rate of TTX association to mutant E945Q is much lower, whereas the off-binding of TTX from D1717Q is much faster, than for WT channels.
Mutation W386Y in the first repeat is fully conservative; mutations E945Q and D1717Q, respectively, in the second and fourth repeat, involve the neutralization of a negative charge; mutation D1426K in the third repeat involves a positive double charge increase.
The four mutations (W386Y, E945Q, D1426K, D1717Q) of the pore region that we studied in this work are illustrated in Fig.
The correction was barely significant for the currents expressed by WT, W386Y, D1426K, and E945Q, whose maximum use-dependent block could be measured for stimulation frequencies lower than 2 Hz; it was substantial, however, in the experiments with mutant D1717Q, which has fast TTX-binding kinetics and shows large UD effects only for stimulation frequencies above 10 Hz.
2 the resting periods were 20 s for mutant D1717Q, 1 min for W386Y and E945Q, 3 min for WT, and 6 min for D1426K.
Notice that the [T] values used in the various experiments vary by more than three orders of magnitude, from 4 nM in the experiment with D1426K to 10 M in the case of mutant D1717Q.
1), with IC(t0 values 5 ranging from 4 nM for D1426K to 11 M for D1717Q.
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
1, yielding the estimates given in the second column of Table 1, which range from 4 nM for D1426K to 11 M for D1717Q .
Peak currents elicited by the various pulses of the train are normalized to those measured with TABLE 1 Summary of the parameters characterizing the affinity and the kinetics of TTX binding to resting and stimulated sodium channel mutants ) IC(t0 (nM) 5 ) IC(s0 (nM) 5 0 (s) 53 7.2 12.0 225 103 16 0.3 0.8 7 6 10 Bi 0.66 0.69 0.67 0.56 0.60 k(0)* (s) off 0.019 0.14 0.083 0.0044 9.71 kon* ( M s) 2.1 1.8 0.038 1.9 2.1 WT* W386Y E945Q D1426K D1717Q 26 247 6.3 3.9 11.3 4 19 0.6 0.6 0.7 103 103 8.9 77 2.2 1.7 4.6 1.2 5 0.2 0.3 0.3 103 103 3 Data are means standard deviation.
5, including measurements on mutant D1717Q that required a more complex analysis discussed Boccaccio et al.
Data for mutant D1717Q are obtained from measurements of the type described in Fig.
f, D1426K; OE, W386Y; , E945Q; F, D1717Q.
3, using the IC(s0 values given in the third column of Table 1, 5 ranging from 1.7 nM for D1426K to 4.6 M for D1717Q.
(B) Similar comparison for 5 the [T] dependence of the time constant, , of the stimulated decay of toxin-free probability, normalized for each phenotype, to the respective estimated upper limit, 0, given in the fourth column of Table 1 and ranging from 103 ms for D1717Q to 225 s for D1426K.
Data for D1717Q are from measurements described in Fig.
5 A versus the logarithm of [T]; they were 236 Biophysical Journal Volume 77 July 1999 FIGURE 6 Time course of the normalized peak response of mutants D1426K (A) and D1717Q (B) to a fixed pulse stimulation during wash-in/ wash-out experiments.
(B) Similar experiment on an oocyte expressing the 1000-fold less TTX-sensitive mutant D1717Q.
6 B shows a wash-in/wash-out experiment on an oocyte expressing the least TTX-sensitive mutant D1717Q, which, as discussed later, shows very fast TTX binding relaxations on a time scale of tens of milliseconds.
Evidently, any extra block possibly induced by a single pulse in mutant D1717Q develops and subsides entirely in less than 0.6 s.
Indeed, we show below that use-dependent relaxations of TTX block also occur in mutant D1717Q, but they are much too fast to be seen with the relatively low frequency of stimulation used in this experiment.
6 B reflects the binding of TTX to resting channels, and data from this and similar experiments with D1717Q are accordingly plotted in Fig.
5 Use-dependent block of mutant D1717Q Finding a constant fraction of blocked D1717Q channels when testing with pulse depolarizations at 0.6-s intervals 4 for the increase of stimulated block upon switching to [T] nM.
Indeed, if the 400) fold increase in the IC(t0 of mutant D1717Q arises primarily 5 from an increase in the rate of TTX dissociation, the relax) ation time of TTX binding to D1717Q at the IC(t0 is ex5 pected to be 400 times shorter than the respective value for WT, falling in the range of tens of milliseconds.
7 illustrates an experiment on an oocyte expressing D1717Q channels and tested with high-frequency repetitive stimulations before and after the addition to the bathing solution of 10 M TTX.
The stimulation protocol consisted of 20 identical pulses of 2 ms to 10 mV separated by a FIGURE 7 Fast use-dependent relaxations of the blockade of D1717Q channels by TTX.
5 to characterize the block by TTX of open D1717Q channels.
As for all of the other phenotypes in this study, the activated state of D1717Q channels is more than twice as sensitive to TTX block than the resting state.
The major distinctive feature of D1717Q appears to be that the kinetics of TTX binding to these channels is 500 times faster than for WT.
As a further support to this conclusion, double-pulse measurements (corrected as above for normal inactivation) show that also for D1717Q the transient extra block induced by a single pulse is biexponential (Fig.
A ) ) simple interpretation of the fair invariance of IC(t0/IC(s0 is 5 5 that our mutations, although changing by several kT units the free energy of the toxin-receptor complex (e.g., an increase of 6kT for D1717Q relative to WT), have little influence on the distance between a bound TTX and a trapped cation and on the probability of cation occupancy of the outermost site in the conduction pore.
The presence off of toxin binding relaxations that can be driven (and measured) by electrical stimulations allowed us to measure these rate constants even when the time constant of the relaxation process was a few tens of milliseconds, as in the case of mutant D1717Q.
The case of E945Q, which has a low TTX sensitivity comparable to that of D1717Q (see Table ) 1), appears quite anomalous.
10097182
full text
S589C
protein
substitution
P37089
true positive
INa at 100 mV was 9.5 2 .3 A for w t, 2.9 0.5 A for S589A, 3.1 0.5 A for S589C, and 1.4 0.4 A for the S589D mut ant.
Coex pression of mut ant subunits s ( S589A, S589C, and S589D) w ith w ild-t ype (w t) and ubun its produced channels that exhibited a sign ificant amilor i d e - s e n s i t i v e K i n w a r d c u r r e n t ( I K) , i n c o n t r a s t t o E N a C w t , which is highly selective for Na over K .
The ion ic per meabilit y profile of the three functional mut ants S589A, S589C, and S589D, is illustrated by macrosc opic I V relationships in the presence of either external Na , Li , or K ions (Fig.
The I V relationship for inward Li current is quite similar for w t and the S589D and S589C mut ants, indicating c onser ved Li over N a s e l e c t i v i t y .
IK INa is only slightly smaller for S589C, but p sign ificantly lower for S589A.
Macrosc opic amiloride-sensitive currents and current ratios at 100 mV I Na, A 3.8 0.5* 0.1* 1.5* 0.7* 2.5 I K I Na 0.06 0.21 0.32 0.01 0.00 0.00 0.01* 0.02* 0.02* 0.01 0.00 0.01 I Li I Na 0.95 1.29 1.42 1.41 2.20 1.47 0.05* 0.04 0.04 0.11 0.2* 0.05 S589A S589C S589D S531A S543A wt 36.0 6.0 1.5 12.1 0.2 27.3 S589A S589A* S589D* S531A S543A wt w t* Amiloride-sensitive currents were measured in solutions c ont ain ing 120 mM Na , Li , or K (see Methods).
S589C, which shows a higher IK INa ratio than S589A, exhibits a sign ificant inward current in the presence of external Rb but not w ith Cs .
R e p l a c e m e n t o f e x t e r n a l N a b y 4 0 m M C a2 y i e l d e d l a r g e amiloride-sensitive inward currents in S589C and S589D Physiolog y: Kellenberger et al .
INa at 100 mV was 29.5 4.0 A for w t, 49.1 3.4 A for S589A, 6.0 0.5 A for S589C, and 4.5 0.9 A for the S589D mut ant.
Fits to the equation Ix INa f (1-[d 2] r)2, where f is a scaling factor, d is the diameter of the nonhydrated ion, and r is the c ylinder radius of the pore, are shown for ENaC w t (solid line), S589A (long dash), S589C (short dash), and S589D (dotted line).
( B ) I V relationship of ENaC w t (E), the S589A (s), S589C (), and S589D (OE) mut ant are shown.
INa at 100 mV was 32.6 14.9 A for w t, 14.5 1.7 A for S589A, 2.9 2.0 A for S589C, and 5.6 0.6 A for the S589D mut ant.
These dat a suggest that S589A is imper meant to Ca2 , and that S589D is more per meant than S589C to Ca2 ions.
5 B c onfir m that ICa of S589D and S589C mut ants is detected essentially at negative potentials where out ward currents (IK t nd I ) are small, and that S589D is more per meant to Ca2 a Na han is S589C.
W i t h 4 0 m M S r2 , small but sign ificant inward currents were detected for S589C, and for the S589D mut ant the ISr INa ratio was 0 0.06 0.01, ISr INa s i m i l a r t o t h e I Ca I Na r a t i o ( I Ca I Na .05 0.01).
N o i n w a r d I Ba c o u l d b e d e t e c t e d w i t h S589C.
Taken together, the ex periments w ith divalent cations show that S589C is per meant to Ca2 and Sr2 , and that a negatively charged amino acid at position 589 is not required for allow ing divalent cations to go through the channel.
Relative macrosc opic amiloride-sensitive currents w ith divalent cations at 100 mV I Mg I Na I Ca I Na 0.01 0.00 0.06 0.01 0.01 0.00 0.01* 0.02 I Sr I Na 0.01 0.01 0.05 0.02 0.00* 0.00* 0.01* 0.00 I Ba I Na 0.01 0.00 0.03 0.01 0.00 0.00 0.00* 0.00 S589A S589C S589D wt 0.00 0.00 0.01 0.02 0.00 0.00* 0.01 0.00 Amiloride-sensitive currents carried by divalent cations relative to I Na are shown.
I Na at 100 mV was 31.7 20.0 A for w t, 12.2 17.7 A for S589A, 3.2 1.7 A for S589C, and 5.9 2.0 A for the S589D mut ant.
These obser vations imply that the pore diameter at the selectiv it y filter is critical for ion discrimination of all the S589 mut ants, for S589C and S589D w ith a high IK INa ratio as well as for S589A, whose relative K per meabilit y is closer to the w t value.
Assuming that the side chain of the 589 residue points toward the pore lumen, enlargement is not ex pected for S589C or S589D mut ations.
Supporting the v iew that the S589 side chain points away f rom the pore lumen is the fact that we have been unable to block S589C channels or to change their af fin it y for amiloride w ith the sulfhydr yl reagents MTSET, MTSEA, or MTSES, suggesting that the S589C side chain is not ac cessible for these reagents.
The divalents Ca2 , Mg2 , Sr2 , and Ba2 block ENaC w t in a volt age-dependent manner (12), and some go through the S589C and S589D mut ants.
S589C has a smaller molecular cutof f c ompared w ith S589D and is less per meant to divalents.
S589A
protein
substitution
P37089
true positive
Two-electrode voltage-clamp rec ordings in Xenopus ooc y tes ex pressing ENaC w t or the S589A, C, or D mut ant are shown.
INa at 100 mV was 9.5 2 .3 A for w t, 2.9 0.5 A for S589A, 3.1 0.5 A for S589C, and 1.4 0.4 A for the S589D mut ant.
Coex pression of mut ant subunits s ( S589A, S589C, and S589D) w ith w ild-t ype (w t) and ubun its produced channels that exhibited a sign ificant amilor i d e - s e n s i t i v e K i n w a r d c u r r e n t ( I K) , i n c o n t r a s t t o E N a C w t , which is highly selective for Na over K .
Amiloride KI values were 0.11 0.02 (n 35), 0.12 0.04 (n 15) for ENaC w t, S589A and ), and 0.13 0.07 M (n S589D, respectively.
The ion ic per meabilit y profile of the three functional mut ants S589A, S589C, and S589D, is illustrated by macrosc opic I V relationships in the presence of either external Na , Li , or K ions (Fig.
With Li ions as charge carrier, inward currents are larger than w ith Na except for S589A, which shows equal currents for Na and Li ions.
IK INa is only slightly smaller for S589C, but p sign ificantly lower for S589A.
Macrosc opic amiloride-sensitive currents and current ratios at 100 mV I Na, A 3.8 0.5* 0.1* 1.5* 0.7* 2.5 I K I Na 0.06 0.21 0.32 0.01 0.00 0.00 0.01* 0.02* 0.02* 0.01 0.00 0.01 I Li I Na 0.95 1.29 1.42 1.41 2.20 1.47 0.05* 0.04 0.04 0.11 0.2* 0.05 S589A S589C S589D S531A S543A wt 36.0 6.0 1.5 12.1 0.2 27.3 S589A S589A* S589D* S531A S543A wt w t* Amiloride-sensitive currents were measured in solutions c ont ain ing 120 mM Na , Li , or K (see Methods).
Patch-clamp rec ordings were per for med on the S589A and S589D mut ants to deter mine the ef fects of the S589 substitutions on single-channel currents.
3, the S589A mut ant behaves ver y similarly to w t in ter ms of single-channel currents and channel gating.
Single-channel Na and Li c onduct ances of w t and the S589A mut ant are identical, indicating a c onser ved Na over Li selectiv it y (Table 2).
The reason for the c discrepanc y bet ween macrosc opic and un it ar y Li and Na urrents w ith S589A and S589D is not k nown, but may be ex plained by changes in channel gating.
Single-channel rec ords of ENaC w t and S589A and S589D mut ant channels.
The S589A mut ant, which has the lowest IK INa ratio, shows inward a currents for Li , Na , and K , and out ward currents w ith Rb and Cs , indicating that it is per meable to K but not to Rb nd Cs .
S589C, which shows a higher IK INa ratio than S589A, exhibits a sign ificant inward current in the presence of external Rb but not w ith Cs .
INa at 100 mV was 29.5 4.0 A for w t, 49.1 3.4 A for S589A, 6.0 0.5 A for S589C, and 4.5 0.9 A for the S589D mut ant.
Fits to the equation Ix INa f (1-[d 2] r)2, where f is a scaling factor, d is the diameter of the nonhydrated ion, and r is the c ylinder radius of the pore, are shown for ENaC w t (solid line), S589A (long dash), S589C (short dash), and S589D (dotted line).
The Cs value for S589A was omitted in the figure for clarit y.
( B ) I V relationship of ENaC w t (E), the S589A (s), S589C (), and S589D (OE) mut ant are shown.
INa at 100 mV was 32.6 14.9 A for w t, 14.5 1.7 A for S589A, 2.9 2.0 A for S589C, and 5.6 0.6 A for the S589D mut ant.
N o a m i l o r i d e - s e n s i t i v e i n w a r d c u r r e n t c o u l d be detected w ith ENaC w t or S589A in 40 mM Ca2 solution.
These dat a suggest that S589A is imper meant to Ca2 , and that S589D is more per meant than S589C to Ca2 ions.
ENaC w t and the S589A mut ant show no amiloride-sensitive ICa in the presence of extracellular Ca2 .
Relative macrosc opic amiloride-sensitive currents w ith divalent cations at 100 mV I Mg I Na I Ca I Na 0.01 0.00 0.06 0.01 0.01 0.00 0.01* 0.02 I Sr I Na 0.01 0.01 0.05 0.02 0.00* 0.00* 0.01* 0.00 I Ba I Na 0.01 0.00 0.03 0.01 0.00 0.00 0.00* 0.00 S589A S589C S589D wt 0.00 0.00 0.01 0.02 0.00 0.00* 0.01 0.00 Amiloride-sensitive currents carried by divalent cations relative to I Na are shown.
I Na at 100 mV was 31.7 20.0 A for w t, 12.2 17.7 A for S589A, 3.2 1.7 A for S589C, and 5.9 2.0 A for the S589D mut ant.
Thus the S589 mut ations increase the channel molecular cutof f to dif ferent extents, the S589D allow ing larger ions to per meate the channel than does the S589A mut ant.
These obser vations imply that the pore diameter at the selectiv it y filter is critical for ion discrimination of all the S589 mut ants, for S589C and S589D w ith a high IK INa ratio as well as for S589A, whose relative K per meabilit y is closer to the w t value.
S589D
protein
substitution
P37089
true positive
( A ) Current traces of ENaC w t and the S589D mut ant in 120 mM K solution.
( B ) Inhibition cur ves of IK (OE) and INa ( ) in the S589D mut ant by amiloride in one ex periment including n ine ooc y tes per c ondition.
INa at 100 mV was 9.5 2 .3 A for w t, 2.9 0.5 A for S589A, 3.1 0.5 A for S589C, and 1.4 0.4 A for the S589D mut ant.
Coex pression of mut ant subunits s ( S589A, S589C, and S589D) w ith w ild-t ype (w t) and ubun its produced channels that exhibited a sign ificant amilor i d e - s e n s i t i v e K i n w a r d c u r r e n t ( I K) , i n c o n t r a s t t o E N a C w t , which is highly selective for Na over K .
Represent ative current traces of ENaC w t and S589D are shown in Fig.
When K replaced Na in the external medium, no amiloridesensitive inward current was measured in ooc y tes ex pressing ENaC w t, whereas a robust IK was detected w ith S589D at negative holding potentials.
To c onfir m that K ions indeed per meate S589D, we have deter mined the sensitiv it y of IK to block ing by amiloride.
In ooc y tes ex pressing S589D, the amiloride inhibition cur ves were identical for inward currents w ith 120-mM external K or Na indicating that the currents are carried by Na or K ions through the amiloride-sensitive S589D channel (Fig.
Amiloride KI values were 0.11 0.02 (n 35), 0.12 0.04 (n 15) for ENaC w t, S589A and ), and 0.13 0.07 M (n S589D, respectively.
The ion ic per meabilit y profile of the three functional mut ants S589A, S589C, and S589D, is illustrated by macrosc opic I V relationships in the presence of either external Na , Li , or K ions (Fig.
The I V relationship for inward Li current is quite similar for w t and the S589D and S589C mut ants, indicating c onser ved Li over N a s e l e c t i v i t y .
The S589D mut ant generated a strongly inwardly p r e c t i f y i n g I K, i n d i c a t i n g a v o l t a g e d e p e n d e n c y o f t h e K er meabilit y.
Macrosc opic amiloride-sensitive currents and current ratios at 100 mV I Na, A 3.8 0.5* 0.1* 1.5* 0.7* 2.5 I K I Na 0.06 0.21 0.32 0.01 0.00 0.00 0.01* 0.02* 0.02* 0.01 0.00 0.01 I Li I Na 0.95 1.29 1.42 1.41 2.20 1.47 0.05* 0.04 0.04 0.11 0.2* 0.05 S589A S589C S589D S531A S543A wt 36.0 6.0 1.5 12.1 0.2 27.3 S589A S589A* S589D* S531A S543A wt w t* Amiloride-sensitive currents were measured in solutions c ont ain ing 120 mM Na , Li , or K (see Methods).
Patch-clamp rec ordings were per for med on the S589A and S589D mut ants to deter mine the ef fects of the S589 substitutions on single-channel currents.
In c ontrast, the S589D mut ation clearly reduces single-channel currents w ith Na and Li .
The single-channel c onduct ance of S589D was lower than that of w t and no dif ferent w ith Na or Li ions (Table 2).
The reason for the c discrepanc y bet ween macrosc opic and un it ar y Li and Na urrents w ith S589A and S589D is not k nown, but may be ex plained by changes in channel gating.
Finally, we have not been able to resolve single-channel K currents through the S589D mut ant, even at highly negative holding potentials, presumably because of channel K c onduct ance being lower than 1 pS.
Our dat a indicate that the high K per meabilit y of the S589D mut ants is associated w ith a decrease in singlechannel c onduct ance for Na and Li ions.
The single-channel c onduct ance of the S589D mut ant remained unchanged when extracellular Na was raised f rom 120 mM to 200 mM (1.7 pS and 1.8 pS, respectively, n 3).
Thus, as for the w t, the un it ar y Na c onduct ance of the S589D mut ant reaches saturation at Na c oncentrations 120 mM, suggesting that , f rom outside-out patches; other dat a are f rom cell-att ached patches.
Single-channel rec ords of ENaC w t and S589A and S589D mut ant channels.
the low un it ar y c onduct ance is not directly related to a major decrease in S589D af fin it y for Na ions.
In c ontrast to the saturation of Na c onduct ance, macrosc opic amiloridesensitive K c onduct ance of S589D mut ants increases linearly w ith the extracellular K c oncentration over the c oncentration range of 5 to 150 mM K , indicating a low af fin it y for K ions (dat a not shown).
Thus in the simult aneous presence of Na and K , the current through S589D is carried mainly by Na ions.
Finally, S589D, which shows the highest IK INa ratio, is per meant to both Rb and Cs ions.
The per meabilit y of the S589D mut ant to group I A Na K Cs .
R e p l a c e m e n t o f e x t e r n a l N a b y 4 0 m M C a2 y i e l d e d l a r g e amiloride-sensitive inward currents in S589C and S589D Physiolog y: Kellenberger et al .
INa at 100 mV was 29.5 4.0 A for w t, 49.1 3.4 A for S589A, 6.0 0.5 A for S589C, and 4.5 0.9 A for the S589D mut ant.
Fits to the equation Ix INa f (1-[d 2] r)2, where f is a scaling factor, d is the diameter of the nonhydrated ion, and r is the c ylinder radius of the pore, are shown for ENaC w t (solid line), S589A (long dash), S589C (short dash), and S589D (dotted line).
( A ) Typical traces of amiloride-sensitive whole-cell currents in BA P TA-injected ooc y tes super fused w ith 40 mM Ca2 solution (see Methods) and ex pressing ENaC w t or the S589D mut ant.
( B ) I V relationship of ENaC w t (E), the S589A (s), S589C (), and S589D (OE) mut ant are shown.
INa at 100 mV was 32.6 14.9 A for w t, 14.5 1.7 A for S589A, 2.9 2.0 A for S589C, and 5.6 0.6 A for the S589D mut ant.
These dat a suggest that S589A is imper meant to Ca2 , and that S589D is more per meant than S589C to Ca2 ions.
In ooc y tes ex pressing S589D, 0.1 M amiloride blocked 79 4 % (n 8) of this inward current, indicating that the amiloride af fin it y of S589D in the presence of external Ca2 is of the same order as in the presence of external Na .
Under these c onditions and in the presence of 40 mM Ca2 , a sign ificant inward ICa c ould still be detected w ith the S589D mut ant (Fig.
5 B c onfir m that ICa of S589D and S589C mut ants is detected essentially at negative potentials where out ward currents (IK t nd I ) are small, and that S589D is more per meant to Ca2 a Na han is S589C.
W i t h 4 0 m M S r2 , small but sign ificant inward currents were detected for S589C, and for the S589D mut ant the ISr INa ratio was 0 0.06 0.01, ISr INa s i m i l a r t o t h e I Ca I Na r a t i o ( I Ca I Na .05 0.01).
With 40 mM external Ba2 , the inward current w ith S589D was smaller than w ith Ca2 , as ex pected for an i o n o f l a r g e r r a d i u s .
The Sr2 Ba2 , supportS589D mut ant is per meant to Ca2 ing the v iew that per meabilit y through S589 mut ants is a function of the ion ic radius of the per meant ion.
Relative macrosc opic amiloride-sensitive currents w ith divalent cations at 100 mV I Mg I Na I Ca I Na 0.01 0.00 0.06 0.01 0.01 0.00 0.01* 0.02 I Sr I Na 0.01 0.01 0.05 0.02 0.00* 0.00* 0.01* 0.00 I Ba I Na 0.01 0.00 0.03 0.01 0.00 0.00 0.00* 0.00 S589A S589C S589D wt 0.00 0.00 0.01 0.02 0.00 0.00* 0.01 0.00 Amiloride-sensitive currents carried by divalent cations relative to I Na are shown.
I Na at 100 mV was 31.7 20.0 A for w t, 12.2 17.7 A for S589A, 3.2 1.7 A for S589C, and 5.9 2.0 A for the S589D mut ant.
Thus the S589 mut ations increase the channel molecular cutof f to dif ferent extents, the S589D allow ing larger ions to per meate the channel than does the S589A mut ant.
These obser vations imply that the pore diameter at the selectiv it y filter is critical for ion discrimination of all the S589 mut ants, for S589C and S589D w ith a high IK INa ratio as well as for S589A, whose relative K per meabilit y is closer to the w t value.
Assuming that the side chain of the 589 residue points toward the pore lumen, enlargement is not ex pected for S589C or S589D mut ations.
Among dif ferent S589 mut ants, the i n c r e a s e o f t h e I K I Na r a t i o p a r a l l e l s a n i n c r e a s e i n t h e molecular diameter cutof f of the channel for per meant cations, the S589D mut ant being per meant to all cations of group I A Na K Cs .
Our obser vation that IK of S589D does not saturate w ith increasing K c oncentration f rom 5 to 150 mM shows that the af fin it y of the S589D mut ant for the per meant K remains low.
The fact that the K af fin it y of mut ant channels remains low indicates that the K per meabilit y of the S589D mut ant does not result f rom the generation of a high-af fin it y binding site for K ions in the channel pore.
The S589D mut ation that makes the pore w ide enough to allow K , Rb , and Cs to go through the channel causes an import ant decrease in singlechannel c onduct ance for smaller ions like Na and Li .
The divalents Ca2 , Mg2 , Sr2 , and Ba2 block ENaC w t in a volt age-dependent manner (12), and some go through the S589C and S589D mut ants.
S589C has a smaller molecular cutof f c ompared w ith S589D and is less per meant to divalents.
In S589D, the per meabilit y to small divalents like Ca2 is higher than per meabilit y to the larger ion Ba2 .
Mg2 has a high c ehydration energ y, and as discussed earlier for the low Na d onduct ance in the S589D mut ation, the energ y c ost for stripping of f the hydration shell of Mg2 is likely to be higher than the interaction energ y of the dehydrated Mg 2 w ith c oordinating residues lin ing the channel pore.
S543A
protein
substitution
P37091
true positive
We found no ev idence for K ( er meable channels w ith c orresponding A la substitutions in S531A) or subunits ( S543A) (Table 1).
Macrosc opic amiloride-sensitive currents and current ratios at 100 mV I Na, A 3.8 0.5* 0.1* 1.5* 0.7* 2.5 I K I Na 0.06 0.21 0.32 0.01 0.00 0.00 0.01* 0.02* 0.02* 0.01 0.00 0.01 I Li I Na 0.95 1.29 1.42 1.41 2.20 1.47 0.05* 0.04 0.04 0.11 0.2* 0.05 S589A S589C S589D S531A S543A wt 36.0 6.0 1.5 12.1 0.2 27.3 S589A S589A* S589D* S531A S543A wt w t* Amiloride-sensitive currents were measured in solutions c ont ain ing 120 mM Na , Li , or K (see Methods).
S589R
protein
substitution
P37089
true positive
Two other S589 mut ations ( S589R and S589W) did not ex press detect able a 8miloride-sensitive currents.
S589W
protein
substitution
P37089
true positive
Two other S589 mut ations ( S589R and S589W) did not ex press detect able a 8miloride-sensitive currents.
S531A
protein
substitution
P37090
true positive
We found no ev idence for K ( er meable channels w ith c orresponding A la substitutions in S531A) or subunits ( S543A) (Table 1).
Macrosc opic amiloride-sensitive currents and current ratios at 100 mV I Na, A 3.8 0.5* 0.1* 1.5* 0.7* 2.5 I K I Na 0.06 0.21 0.32 0.01 0.00 0.00 0.01* 0.02* 0.02* 0.01 0.00 0.01 I Li I Na 0.95 1.29 1.42 1.41 2.20 1.47 0.05* 0.04 0.04 0.11 0.2* 0.05 S589A S589C S589D S531A S543A wt 36.0 6.0 1.5 12.1 0.2 27.3 S589A S589A* S589D* S531A S543A wt w t* Amiloride-sensitive currents were measured in solutions c ont ain ing 120 mM Na , Li , or K (see Methods).
12695533
full text
Y652A
protein
substitution
true positive
Q12809
Mutations of Y652 eliminated (Y652F) or reversed (Y652A) the voltage dependence of HERG channel block by quinidine and quinine.
However, similar changes in the voltage-dependent profile for block of Y652F or Y652A HERG channels were observed with vesnarinone, a cardiotonic drug that is uncharged at physiological pH.
Here, we also examined the effects of the uncharged drug vesnarinone on Y652A and Y652F HERG channels to determine whether a drug must possess an ionizable N atom to block HERG channels in a voltage-dependent manner.
Although the block of wild-type and Y652F HERG channels by vesnarinone, an uncharged drug, was relatively insensitive to voltage, block of Y652A HERG was surprisingly decreased at greater depolarized potentials, similar to the charged quinolines.
Point mutations were introduced into HERG (V625A, Y652A, Y652F, F656A) in the pSP64 plasmid expression vector (Promega, Madison, WI), as described previously (Mitcheson et al., 2000).
Therefore, we determined the concentration-effect relationship for quinidine on V625A, Y652A, and F656A HERG channels and compared the potency for block with that of the WT HERG channel.
Peak tail current was measured at 70 mV after a 4-s pulse to 0 mV for WT, V625A, and Y652A HERG channels.
The effect of 10 M quinidine on WT, V625A, and Y652A HERG channel current is shown in Fig.
The IC50 values were 4.6 1.2 M for WT, 17.5 1.9 M for V625A, and 16 1.7 M for Y652A HERG (Fig.
A through C, superimposed traces of WT HERG (A), V625 HERG (B), and Y652A HERG (C) currents elicited by the application of depolarizing pulses to 0 mV before and after exposure to 10 M quinidine.
The IC50 value was 4.6 1.2 M for WT, 1.7 M for Y652A (n 5 for each 17.5 1.9 M for V625A, and 16 group).
However, Y652A channels were 500 times less sensitive to chloroquine and the V625A mutation had no effect on potency of block.
We reported previously that the voltage dependence for block of HERG channels by chloroquine was reversed by the Y652A mutation (Sanchez-Chapula et al., 2002).
The effect of quinidine on Y652A HERG current elicited at test potentials of 40 and 20 mV in the same cell is shown in Fig.
Quinidine blocked Y652A HERG current more effectively at 40 mV than at 20 mV, and the apparent rate of deactivation was faster in the presence of drug.
Voltage-dependent block of Y652A HERG currents by quinidine.
E, time constants for the onset of block and unblock of Y652A HERG current by quinidine plotted as a function of the test potential (Vt).
F, fractional block of Y652A HERG currents by 10 M quinidine plotted as a function of Vt.
Steady-state block of Y652A HERG was also voltage-dependent and varied from 0.7 at 50 mV to 0.04 at 40 mV (Fig.
Thus, quinidine blocked HERG channels only after opening the activation gate, and in opposition to results observed with WT HERG, the block of Y652A HERG decreased with increasing membrane depolarization.
Also in opposition to the WT HERG results, quinidine shifted the voltage dependence of Y652A HERG channels to more positive potentials (Fig.
Similar to rates obtained with WT and Y652A HERG channels, the rate for the onset of block of Y652F channels was faster at more depolarized potentials (Fig.
However, unlike Y652A HERG (Fig.
We also determined the effects of quinine, a stereoisomer of quinidine, on WT, Y652A, and Y652F HERG channels.
7, A and D), blocked Y652A HERG less at depolarized potentials (Fig.
Effect of quinidine on the voltage dependence of Y652A and Y652F HERG channel activation.
A, effect of quinidine on the isochronal activation curves for Y652A HERG.
8, A and D), a dramatically less block of Y652A HERG at depolarized potentials (Fig.
A through C, superimposed traces of WT (A), Y652A (B), and Y652F (C) HERG currents elicited during the application of depolarizing pulses to 40 and 20 mV and upon repolarization to 70 mV before and after exposure to 100 M quinine.
D through F, fractional block of WT, Y652A, and Y652F HERG currents plotted as a function of the test potential (Vt).
A to C, Superimposed traces of WT (A), Y652A (B), and Y652F (C) HERG currents elicited during the application of depolarizing pulses to 40 and 20 mV and upon repolarization to 70 mV before and after exposure to 30 M vesnarinone.
D through F, Fractional block of WT, Y652A, and Y652F HERG currents plotted as a function of test potential (Vt).
In contrast, block of Y652A HERG current by these drugs was diminished by increased depolarization, whereas block of Y652F HERG current was relatively insensitive to voltage.
Thus, substitution of a phenyl with a benzyl moiety (Y652F) eliminated the voltage dependence of HERG block, whereas substitution with a methyl group (Y652A) reversed the voltage dependence of the block.
However, our finding that block of Y652A channels was also voltagedependent for vesnarinone, an uncharged drug, indicates that cation- interactions are not required for voltagedependent block of HERG channels by all drugs.
V625A
protein
substitution
true positive
Q12809
Point mutations were introduced into HERG (V625A, Y652A, Y652F, F656A) in the pSP64 plasmid expression vector (Promega, Madison, WI), as described previously (Mitcheson et al., 2000).
Therefore, we determined the concentration-effect relationship for quinidine on V625A, Y652A, and F656A HERG channels and compared the potency for block with that of the WT HERG channel.
Peak tail current was measured at 70 mV after a 4-s pulse to 0 mV for WT, V625A, and Y652A HERG channels.
The effect of 10 M quinidine on WT, V625A, and Y652A HERG channel current is shown in Fig.
As reported previously (Mitcheson et al., 2000), the tail currents for V625A HERG channels were inward at 70 mV because of a change in ion selectivity.
The IC50 values were 4.6 1.2 M for WT, 17.5 1.9 M for V625A, and 16 1.7 M for Y652A HERG (Fig.
The IC50 value was 4.6 1.2 M for WT, 1.7 M for Y652A (n 5 for each 17.5 1.9 M for V625A, and 16 group).
However, Y652A channels were 500 times less sensitive to chloroquine and the V625A mutation had no effect on potency of block.
In contrast, we found that V625A HERG channels were 3.8-fold less sensitive to block by quinidine than were WT HERG channels.
F656A
protein
substitution
true positive
Q12809
Point mutations were introduced into HERG (V625A, Y652A, Y652F, F656A) in the pSP64 plasmid expression vector (Promega, Madison, WI), as described previously (Mitcheson et al., 2000).
Therefore, we determined the concentration-effect relationship for quinidine on V625A, Y652A, and F656A HERG channels and compared the potency for block with that of the WT HERG channel.
To increase the amplitude of poorly expressing F656A mutant channels, tail currents were recorded at Fig.
Using this p 1rotocol, the IC50 was 5.2 0.9 M for WT current and 650 16 M for F656A HERG current (Fig.
We reported previously that the block of F656A channels by chloroquine was also greatly (approximately 1000 times) reduced (Sanchez-Chapula et al., 2002).
The F656A mutation caused a much larger shift in the IC50 for chloroquine, 500-fold (Sanchez-Chapula et al., 2002).
Y652F
protein
substitution
true positive
Q12809
Mutations of Y652 eliminated (Y652F) or reversed (Y652A) the voltage dependence of HERG channel block by quinidine and quinine.
However, similar changes in the voltage-dependent profile for block of Y652F or Y652A HERG channels were observed with vesnarinone, a cardiotonic drug that is uncharged at physiological pH.
Here, we also examined the effects of the uncharged drug vesnarinone on Y652A and Y652F HERG channels to determine whether a drug must possess an ionizable N atom to block HERG channels in a voltage-dependent manner.
Although the block of wild-type and Y652F HERG channels by vesnarinone, an uncharged drug, was relatively insensitive to voltage, block of Y652A HERG was surprisingly decreased at greater depolarized potentials, similar to the charged quinolines.
Point mutations were introduced into HERG (V625A, Y652A, Y652F, F656A) in the pSP64 plasmid expression vector (Promega, Madison, WI), as described previously (Mitcheson et al., 2000).
Like chloroquine, quinidine block of Y652F HERG channels was also weakly voltage-dependent (Fig.
The rate of Y652F HERG channel deactivation was only slightly altered by quinidine, but the rate was best fit with a single exponential function rather than the biexponential function required for the WT HERG current.
Similar to rates obtained with WT and Y652A HERG channels, the rate for the onset of block of Y652F channels was faster at more depolarized potentials (Fig.
4D), block of Y652F HERG channels by quinidine at 20 mV was not followed by a recovery from block (Fig.
6D), and the half-point for activation of Y652F HERG was only slightly affected (Fig.
Steady-state fractional block of Y652F channels was weakly voltage-dependent at potentials between 30 and 10 mV (Fig.
We also determined the effects of quinine, a stereoisomer of quinidine, on WT, Y652A, and Y652F HERG channels.
7, B and E), and blocked Y652F HERG independent of voltage (Fig.
Effect of quinidine on the voltage dependence of Y652A and Y652F HERG channel activation.
B, the effect of quinidine on the isochronal activation curves for Y652F HERG.
8, B and E), and a slightly less block of Y652F HERG at depolarized potentials (Fig.
Voltage-dependent block of Y652F HERG currents by quinidine.
E, time constants for onset of block of Y652F HERG current by quinidine plotted as a function of the test potential (Vt).
F, fractional block of Y652F HERG currents plotted as a function of Vt.
A through C, superimposed traces of WT (A), Y652A (B), and Y652F (C) HERG currents elicited during the application of depolarizing pulses to 40 and 20 mV and upon repolarization to 70 mV before and after exposure to 100 M quinine.
D through F, fractional block of WT, Y652A, and Y652F HERG currents plotted as a function of the test potential (Vt).
A to C, Superimposed traces of WT (A), Y652A (B), and Y652F (C) HERG currents elicited during the application of depolarizing pulses to 40 and 20 mV and upon repolarization to 70 mV before and after exposure to 30 M vesnarinone.
D through F, Fractional block of WT, Y652A, and Y652F HERG currents plotted as a function of test potential (Vt).
In contrast, block of Y652A HERG current by these drugs was diminished by increased depolarization, whereas block of Y652F HERG current was relatively insensitive to voltage.
Thus, substitution of a phenyl with a benzyl moiety (Y652F) eliminated the voltage dependence of HERG block, whereas substitution with a methyl group (Y652A) reversed the voltage dependence of the block.
11226332
full text
E4032A
protein
substitution
true positive
P11716
Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine James D.
We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype.
When expressed in 1B5 (RyR null dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads.
However, treatment of E4032A-expressing cells with 200 500 M ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists.
Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine.
In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 s) gating events and insensitivity to Ca2 .
In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.
One of these point mut ations per for med in an RyR3 c ontext, E3885A (which is analogous to E4032A in RyR1), decreases sensitiv it y to calcium activation by 10,000-fold (14).
In the present study, we characterized E4032A RyR1 expressed in the RyR null dyspedic 1B5 myogen ic cell line (18).
A finding which clarifies the molecular mechan ism of r yanodine action reveals that dyspedic myotubes expressing E4032A regain their abilit y to respond to depolarizing potentials, caf feine, and 4-chloro-m-cresol af ter the addition of micromolar r yanodine.
Ryanodine also restores single channel activ it y, demonstrating that unlike w ild-t ype RyR1 (w tRyR1), micromolar c oncentrations of this alk aloid do not persistently block the activ it y of the E4032A channels.
The oligonucleotides for the E4032A mut ation are as follows: for ward, 5 GTCCCTACTGGCAGGGA ACGTGGT-3 and reverse, 5 CCACGT TCCCTGCCAGTAGGGACA-3 .
The XhoI (12018)-StuI (12224) f ragment was removed f rom the PCR product and was used to replace the c orresponding w t region to for m the full-length E4032A-RyR1 cDNA.
Herpes simplex v ir us-1 v irions (21) c ont ain ing the Construction of E4032A RyR1 cDNA.
The point mut ation E4032A rec onstituted into BLM were per for med by using Cs as charge carrier as described prev iously (19).
Membrane vesicles c ont aining RyR channels for rec onstitution ex periments were isolated f rom 1B5 myotubes ex pressing E4032A as described above, that had been either ex posed to 200 M r yanodine for 24 h or lef t untreated.
In separate ex periments, heav y SR membranes c ont ain ing E4032A isolated f rom cells not ex posed to r yanodine during culture were treated directly w ith 200 M r yanodine in the test tube for 30 min at 37C before rec onstitution in BLM.
high-af fin it y sites on SR prepared f rom 1B5 myotubes ex pressing either w tRyR1 or E4032A was per for med as prev iously described (18) in the absence or presence of 20 mM caf feine and 1 mM adenosine 5 -[ , -methylene]triphosphate (A MP-PCP; a nonhydrolyzable AT P analog).
Measurements of single E4032A channels E4032A-ex pressing 1B5 myotubes were fixed in 3.5% glut araldehyde in 0.1 M sodium phosphate buf fer (pH 7.4) at room temperature.
cDNA enc oding either w tRyR1 or E4032A-RyR1 were added to dif ferentiated 1B5 myotubes at a c oncentration of 3 105 infectious un its (IU) ml.
myotubes infected w ith E4032A cDNA-c ont ain ing v irions were prepared as described prev iously (18, 19), and subsequently loaded onto a sucrose g radient c onsisting of layers of 10%, 27%, and 45% (w t wt) sucrose in 10 mM Hepes (pH 7.4).
Cr ude membrane homogenates f rom Results The mut ant E4032A RyR1 cDNA was introduced into 1B5 dyspedic myotubes by using the p-HSV amplic on system (21).
1B5 myotubes ef ficiently ex press E4032A RyR1 (which is the ex pected size by Western blot analysis; Fig.
In f reeze f racture replicas, the sur face membrane of E4032A-infected cells shows clusters of dihydropy ridine receptor (DHPR) particles grouped into tetrads (Fig.
24), thus indicating that the E4032A mut ant can associate w ith the DHPR in a manner similar to w tRyR1.
The functional phenot ype of the E4032A RyR1 mut ation was deter mined by ex pressing this mut ated protein in 1B5 myotubes and examin ing functional responses by using Ca2 imaging techn iques w ith the Ca2 -sensitive dye Fura-2.
The number of RyR-transduced 1B5 myotubes responding to RyR agon ists was used as a semiquantit ative measure of E4032A function.
1B5 myotubes ex pressing E4032A generally failed to exhibit excit ation c ontraction c oupling (Fig.
The lack of responsiveness of the E4032A mut ant extended to direct cellper meant modulators of w tRyR1, including 40 mM caf feine (13% responding) and 0.5 mM 4-chloro-m-cresol (6.4% responding).
Commensurate w ith the lack of E4032A function were ver y low, but clearly discernable, levels of high-af fin it y (10 nM) [3H]r yanodine-binding sites in membrane preparations isolated f rom E4032A-ex pressing 1B5 cells (Fig.
RyR1 carrying the E4032A mutation is properly expressed in 1B5 myotubes.
1B5 cells grown in collagen-coated 35-mm dishes were infected with either RyR1 or E4032A cDNA-containing herpes simplex virus amplicon virions at 3 105 IU ml.
(A) Western blot of 1B5 myotube preparations expressing E4032A-RyR1 (lane 1, 20 g), 1B5 null myotubes not transduced with cDNA (lane 2, 20 g), and 1B5 myotubes expressing wtRyR1 (lane 4, 20 g).
(B) The intracellular distribution of E4032A expressed in 1B5 myotubes was examined by using immunocytochemistry as described in Materials and Methods.
E4032A was properly targeted to junctional domains at the fiber periphery as indicated by the punctate appearance of the immunolabeling pattern which was indistinguishable from the pattern obtained when wtRyR1 was expressed in 1B5 cells (C).
(D and E) E4032A- and wtRyR1expressing 1B5 myotubes were examined for DHPR tetrad formation by freezefracture electron microscopy.
E4032A channels are largely unresponsive to activation by RyR agonists.
(A) 1B5 cells grown in collagen-coated 72-well microtiter plates (Terasaki format) were infected with either E4032A or wtRyR1 cDNA containing herpes simplex virions at 3 105 IU ml.
The change in cytoplasmic calcium (as indicated by a change in F340 F380 ratio for Fura-2) in response to 40 mM KCl, 40 mM caffeine, 0.5 mM 4-chloro-m-cresol, or 2 M ionomycin for either a wtRyR1-expressing myotube (upper trace) or E4032Aexpressing myotube (lower trace) is indicated.
50 sec.) (B) E4032A RyR1 shows largely reduced high-affinity [3H]ryanodine binding.
wtRyR1 or E4032A RyR1-expressing 1B5 membrane preparations were incubated at 37C for 3 h in a buffer containing 250 mM KCl, 15 mM NaCl, 20 mM Pipes (pH 7.4), and 10 nM [3H]ryanodine.
(C) Single channel measurements of isolated E4032A channels reconstituted in BLM were conducted as described in Materials and Methods.
Isolated E4032A channels give rise to infrequent gating transitions from the closed to fully open state in the presence of cis 1 mM calcium and 2 mM ATP.
Lack of RyR-dependent functional responses of myotubes ex pressing E4032A and low oc cupanc y of nanomolar [3H]r yanodine suggested that E4032A channels were likely to exhibit inherently low open probabilit y.
To address this hypothesis, the same SR vesicles used for binding studies were fused w ith artificial BLM, and the single-channel characteristics of E4032A RyR were studied.
E4032A channels displayed inf requent gating transitions and an extremely low open probabilit y when c ompared w ith w tRyR1 (Fig.
E4032A channels were also largely unresponsive to activation either by cis (c y toplasmic) calcium bet ween 7 M and 100 M in the presence of 1 mM AT P or the phar mac ological agon ist caf feine.
14), although E4032A RyR1 activit y remained low (Po 0.0045 0.0014, mean SE, n 11 channels, Fig.
A lthough E4032A channels possess int act high-af fin it y binding domains for r yanodine, the apparent af fin it y for alk aloidinduced modifications of function should be sign ificantly reduced as a direct result of the inherently low Po c ontributed by the mut ation.
To test this hypothesis, we examined the ef fect of 500 M r yanodine on 1B5 cells ex pressing the E4032A mut ant protein.
Interestingly, we enc ountered an unex pected result: treatment of E4032A-ex pressing 1B5 myotubes w ith 500 M r yanodine for 30 min restored the function of the mut ated Fessenden et al.
E4032A RyR1 (Fig.
In these ex periments, we examined caf feine responses of E4032A-transduced cells before and af ter r yanodine application and c orrelated the presence of a functional response in an indiv idual cell w ith the ex pression of E4032A protein in the same cell.
The cells were first tested functionally and subsequently identified by using immunoc y tochemistr y to c onfir m that cells show ing Ca2 transients af ter r yanodine treatment ex pressed E4032A RyR1.
Our results indicated that r yanodine treatment recr uits E4032A-ex pressing cells to bec ome responsive to RyR agon ists (Fig.
In the represent ative field shown, only 2 of 25 cells identified as E4032A-ex pressing responded to an in itial challenge w ith 40 mM caf feine (Fig.
However, af ter a 30 min treatment w ith 500 M r yanodine, 14 of 25 E4032A-ex pressing cells were responsive to a sec ond 40 mM caf feine application (Fig.
The r yanodine application by itself did not elicit any increase in [Ca2 ]i in E4032A-ex pressing cells, although subsequent responses to 40 mM caf feine were robust (Fig.
To c onfir m that micromolar r yanodine restores functional activ it y of E4032A, we tested the abilit y of the r yanodine-treated E4032A-ex pressing cells to support excit ation c ontraction c oupling and respond to RyR agon ists.
Af ter a 30 min to 24 h r yanodine incubation followed by removal of the alk aloid, the percent age of E4032A-infected cells responding to 40 mM KCl, 40 mM caf feine, and 0.5 mM chloro-m-cresol increased to 27%, 37%, and 38%, respectively (n 412 cells examined; Fig.
Ryanodine restores E4032A activity in 1B5 myotubes.
1B5 myotubes expressing E4032A were initially examined for functional responses to RyR agonists.
E4032A expression was determined by immunocytochemical analysis after methanol fixation.
A and B show the same field of cells after immunostaining using 34C antibody to reveal 25 cells expressing E4032A.
(A) Only 2 of these E4032A-expressing cells responded to 40 mM caffeine (arrows).
(B) After addition of 500 M ryanodine for 30 min, 12 additional E4032A-expressing cells responded to a subsequent application of 40 mM caffeine (arrowheads).
Ryanodine-pretreated E4032A channels become responsive to RyR agonists.
(A) In an E4032A-expressing 1B5 myotube pretreated with 500 M ryanodine for 30 min, responses to 40 mM KCl, 40 mM caffeine and 0.5 mM 4-chloro-m-cresol are restored.
50 sec.) (B) The degree of restoration of E4032A activity by ryanodine is indicated.
A small percentage of the total number of cells examined for changes in calcium respond to RyR agonists in dishes containing untreated E4032A-expresssing 1B5 cells (clear bar).
(Inset): E4032A-expressing 1B5 myotubes were incubated with increasing concentrations of ryanodine for 24 h.
(C) Ryanodine restores skeletal-type excitation contraction coupling of E4032A.
Addition of 40 mM KCl (black bar) to an E4032A-expressing cell pretreated with 500 M ryanodine for 24 h produced calcium transients in both the presence and absence of extracellular calcium, indicating a functional interaction between RyR and DHPR.
60 sec.) (D) E4032A activity can be restored by ryanodine in single-channel studies.
Single-channel measurements of E4032A channels reconstituted in BLM were conducted as described in Materials and Methods.
E4032A channels isolated from 1B5 cells pretreated for 24 h with 200 M ryanodine are active at 7 M calcium cis (upper trace).
In E4032A channels pretreated with ryanodine, channel activity depended on the level of calcium in the cis chamber of the bilayer, since lowering the level of calcium to 3.4 nM ( 1 min before recording) fully inactivated the channel (lower trace).
Ryanodine restored responsiveness of E4032A-ex pressing 1 c e l l s t o c a f f e i n e i n a d o s e d e p e n d e n t m a n n e r w i t h a n E C 50 65 M (Fig.
This restoration of E4032A activ it y c ontinued to be obser ved 90 min af ter the r yanodine was removed by extensive washing.
When E4032A-ex pressing 1B5 myotubes were pretreated w ith 200 M r yanodine for 24 h, the rec onstituted channels became sign ificantly more active in the presence of 7 M Ca2 (cis) w ith multiple subst ates, of which the 1 4 st ate predominated (Fig.
The subst ates where Po averaged 0.3 of the r yanodine-treated E4032A channels were not obser ved in the c ontrol (untreated) E4032A channels.
In the r yanodinetreated E4032A channels, gating behav ior depended on the Ca2 2868 www.pnas.org cgi doi 10.1073 pnas.041608898 oncentration on the cis face of the channel (Fig.
Rec onstitution of E4032A channel activ it y by r yanodine c ould also be achieved by treating E4032A-c ont ain ing SR vesicles for 30 min w ith 200 M r yanodine.
These results demonstrate that r yanodine treatment can reverse the phenot ype of the E4032A mut ation, thus enabling the channel to bec ome responsive to activation.
To deter mine whether r yanodine can disr upt calcium release in 1B5 myotubes ex pressing E4032A, we tested the ef fect of caf feine added together w ith r yanodine (Fig.
Addition of 40 mM caf feine to a r yanodine-pretreated E4032A-ex pressing myotube produced a calcium transient.
Calcium transients from ryanodine-pretreated E4032A-expressing 1B5 cells are not affected by ryanodine.
1B5 myotubes infected with viruses containing E4032A or wtRyR1 cDNA (3 105 IU ml) were imaged for calcium as described in Materials and Methods.
(A) Pretreatment of E4032A with 500 M ryanodine for 24 h restored responsiveness to 40 mM caffeine (clear bar).
Taken together, these results indicate that r yanodine does not alter Ca2 release through the E4032A channel, but instead restores its abilit y to respond to k nown stimuli of the RyR.
Discussion Our results indicate that the E4032A mut ation severely c ompromises channel-gating activ it y.
This finding is c onsistent w ith prev ious studies on E4032A ex pressed in HEK-293 cells (15) as well as studies on the c orresponding mut ation per for med in RyR3 (E3885A; ref.
In this regard, E4032A RyR1 may c ont ain a localized c onfor mational change that c ould st abilize the closed st ate, dest abilize the open st ate, or af fect both, leading to an energetically unfavorable closedto-open channel transition (Fig.
The E4032A mut ation does not seem to af fect RyRDHPR str uctural interactions.
Proposed model showing the interaction between ryanodine and either the wtRyR1 or the E4032A RyR mutant.
By contrast, E4032A exhibits a large energy barrier that is not affected by Ca2 , caffeine, and AMP-PCP, singly or in combination.
A high concentration of ryanodine promotes sequential binding to allosterically coupled sites on E4032A, but the outcome is a dramatic decrease in free energy associated with channel gating between closed (C ) and open (O ) states in the ryanodine-modified E4032A RyR1.
Interestingly, RyR1 carr ying the E4032A mut ation est ablishes this link although functional responses to membrane depolarization are absent, thus indicating that this loss of function is not caused by disr uption of the RyR-DHPR interaction.
T h e v e r y h i g h c o n c e n t r a t i o n ( E C 50 165 M) of r yanodine needed to restore functional responses of E4032A RyR w ithin myotubes and at the level of single channels can be ex plained by the extremely low oc currence of E4032A channel transitions to the open st ate.
A likely mechan ism is that E4032A introduces a large energ y barrier associated w ith channel transitions f rom closed to open (Fig.
A n import ant finding is that once E4032A is oc cupied by r yanodine, possibly at low-af fin it y sites, functionalit y is essentially restored.
Considering that high c oncentrations of r yanodine produce c omplex changes in channel c onfor mation ultimately leading to persistent changes in w tRyR1 function (8, 9), it is not unreasonable to suggest that the binding of this molecule to an RyR1 possessing an altered c onfor mational topolog y c ould potentially reverse energ y barriers to gating inf licted by the E4032A mut ation (Fig.
Our results suggest that r yanodine may not need to remain bound to restore E4032A activ it y because this activ it y persists even 90 min af ter r yanodine has been removed by extensive washing.
However, the possibilit y still ex ists that r yanodine may remain bound to E4032A RyR1 because of the extremely slow of f-rates of the ligand f rom high-af fin it y sites af ter low-af fin it y sites are oc cupied by r yanodine (9, 27).
The ef fects of r yanodine on E4032A are in c ontrast to its ef fects on w tRyR1.
However, our work w ith the restored E4032A argues 1.
Thus, irreversible ef fects generally attributed to r yanodine binding to low-af fin it y sites are not present for the restored E4032A channel.
One possible interpret ation is that low af fin it y interaction bet ween r yanodine and E4032A relieves energetic barriers associated w ith channel gating by induced allosterism.
A n alternative hypothesis--that the E4032A mut ation directly disr upts the binding site of r yanodine necessar y for channel oc clusion--seems unlikely because photoaf fin it y and tr yptic digest studies have localized high-af fin it y r yanodine binding bet ween residues 4475 and the C ter minus of the protein (16, 17).
Our binding dat a, which show the discernable, although reduced, high-af fin it y binding of [3H]r yanodine to the E4032A mut ant, also suggest that this site is int act.
Thus, our results w ith the E4032A mut ant channel suggest that r yanodine does not act as a pore blocker but instead, that r yanodine binding sites reside outside of the per meation pore, and that r yanodine binding to these sites has allosteric ef fects on calcium per meabilit y.
Chen for prov iding us w ith the E4032A mut ant cDNA and Dr.
E3885A
protein
substitution
true negative
One of these point mut ations per for med in an RyR3 c ontext, E3885A (which is analogous to E4032A in RyR1), decreases sensitiv it y to calcium activation by 10,000-fold (14).
The highest Po values were obt ained in the presence of 1 to 2 mM Ca2 , 2 mM AT P, or 2 mM caf feine (a c ondition which sign ificantly increased the Po of RyR3 E3885A channels; ref.
This finding is c onsistent w ith prev ious studies on E4032A ex pressed in HEK-293 cells (15) as well as studies on the c orresponding mut ation per for med in RyR3 (E3885A; ref.
12928313
full text
H282D
protein
substitution
true positive
P37089
Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type -ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel.
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
Channels formed by mutations W278G, R280G, and H282D in -ENaC are more sensitive to amiloride than are wild-type ENaC.
Although the K0.5 for amiloride of wild-type -ENaC is 0.029 M, t h e K 0.5 v a l u e s f o r a m i l o r i d e o f t h e s e m u t a t i o n s a r e 0.006 M for W278G and R280G and 0.022 M for H282D.
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
On the basis of data from singlechannel analysis and determination of K0.5 for amiloride, most of the mutants fall away from the line, except possibly Y279A and H282D.
Previous work showed that deletion of this region as a whole ( 278283), as well as mutations of individual residues, R280G, H282D, and H282R, alters amiloride binding to the channel (12).
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
The data suggest that the histidine, arginine, and tryptophan residues interact with amiloride on the basis of the differences observed with the H282D/R, R280G/K, and W278G/A mutations and the level of disruption to amiloride binding caused by channels made from -subunits with these mutations.
The deletion of this region, 278283, and point mutations, W278G, R280G, F281A, and H282D, caused a change in the amiloride-blocking rate of the channel.
Y279A
protein
substitution
true positive
P37089
Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type -ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel.
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
Two of the mutants, F281A and Y279A, which produced the most dramatic effects, caused a clear change in the way the channel transitioned from the open state to the closed state compared with wild-type channels.
The mean closed times for 278283, W278G, Y279A, and R280G were larger than those of wild-type (high-Po) channels ( closed 0.031 s).
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
ride include Y279A, F281A, and Y283A, with K0.5 values for amiloride of 0.22, 0.26, and 0.63 M, respectively (Table 4).
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
Mean K0.5 values for wild-type, W278G/A, Y279A/F, and R280G/K channels formed from -ENaC subunits alone expressed in CHO cells.
Dose-response curves of effects of amiloride on the relative change in Po for channels formed from wild-type -ENaC or -ENaC mutations R280G/K, W278G/A, and Y279A/F.
AJP-Renal Physiol VOL WT R280G R280K W278G W278A Y279A Y279F 9 2 3 7 3 9 3 0.029 0.006 0.078 0.006 0.007 0.0004* 0.0004* 0.001* 1* 0.22 0.056* 0.03 0.001* Values are means SE; n, number of cells.
On the basis of data from singlechannel analysis and determination of K0.5 for amiloride, most of the mutants fall away from the line, except possibly Y279A and H282D.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
The closed times for 278283, W278G, Y279A, and R280G were longer than those for wild-type high-Po channels.
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
Y279F
protein
substitution
true positive
P37089
Additional mutations, W278A, Y279F, and R280K, were generated via PCR using primers across the mutation site and subcloning the product back into a wild-type -rENaC/pCDNA3 backbone.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
These new mutations, W278A, Y279F, and R280K, were expressed in CHO cells and compared with wild-type -ENaC and the original mutations generated at these positions (Figs.
The restoration of amiloride sensitivity to wild-type levels by Y279F and R280K mutant channels supports the roles of these residues in the overall binding of amiloride to the wild-type channel.
AJP-Renal Physiol VOL WT R280G R280K W278G W278A Y279A Y279F 9 2 3 7 3 9 3 0.029 0.006 0.078 0.006 0.007 0.0004* 0.0004* 0.001* 1* 0.22 0.056* 0.03 0.001* Values are means SE; n, number of cells.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
R280G
protein
substitution
true positive
P37089
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
The mean closed times for 278283, W278G, Y279A, and R280G were larger than those of wild-type (high-Po) channels ( closed 0.031 s).
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
Error bars, SE; n 3, except for R280G, where n 2.
Channels formed by mutations W278G, R280G, and H282D in -ENaC are more sensitive to amiloride than are wild-type ENaC.
Although the K0.5 for amiloride of wild-type -ENaC is 0.029 M, t h e K 0.5 v a l u e s f o r a m i l o r i d e o f t h e s e m u t a t i o n s a r e 0.006 M for W278G and R280G and 0.022 M for H282D.
Error bars, SE; n 3, except for R280G, where n 2.
Error bars, SE; n 3, except for R280G, where n 2.
Error bars, SE; n 3, except R280G, where n 2.
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
Mean K0.5 values for wild-type, W278G/A, Y279A/F, and R280G/K channels formed from -ENaC subunits alone expressed in CHO cells.
Error bars, SE; n 3, except for R280G, where n 2.
Dose-response curves of effects of amiloride on the relative change in Po for channels formed from wild-type -ENaC or -ENaC mutations R280G/K, W278G/A, and Y279A/F.
AJP-Renal Physiol VOL WT R280G R280K W278G W278A Y279A Y279F 9 2 3 7 3 9 3 0.029 0.006 0.078 0.006 0.007 0.0004* 0.0004* 0.001* 1* 0.22 0.056* 0.03 0.001* Values are means SE; n, number of cells.
Previous work showed that deletion of this region as a whole ( 278283), as well as mutations of individual residues, R280G, H282D, and H282R, alters amiloride binding to the channel (12).
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
The closed times for 278283, W278G, Y279A, and R280G were longer than those for wild-type high-Po channels.
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
The data suggest that the histidine, arginine, and tryptophan residues interact with amiloride on the basis of the differences observed with the H282D/R, R280G/K, and W278G/A mutations and the level of disruption to amiloride binding caused by channels made from -subunits with these mutations.
The deletion of this region, 278283, and point mutations, W278G, R280G, F281A, and H282D, caused a change in the amiloride-blocking rate of the channel.
W278G
protein
substitution
true positive
P37089
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
The mean closed times for 278283, W278G, Y279A, and R280G were larger than those of wild-type (high-Po) channels ( closed 0.031 s).
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
Channels formed by mutations W278G, R280G, and H282D in -ENaC are more sensitive to amiloride than are wild-type ENaC.
Although the K0.5 for amiloride of wild-type -ENaC is 0.029 M, t h e K 0.5 v a l u e s f o r a m i l o r i d e o f t h e s e m u t a t i o n s a r e 0.006 M for W278G and R280G and 0.022 M for H282D.
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
Mean K0.5 values for wild-type, W278G/A, Y279A/F, and R280G/K channels formed from -ENaC subunits alone expressed in CHO cells.
Dose-response curves of effects of amiloride on the relative change in Po for channels formed from wild-type -ENaC or -ENaC mutations R280G/K, W278G/A, and Y279A/F.
AJP-Renal Physiol VOL WT R280G R280K W278G W278A Y279A Y279F 9 2 3 7 3 9 3 0.029 0.006 0.078 0.006 0.007 0.0004* 0.0004* 0.001* 1* 0.22 0.056* 0.03 0.001* Values are means SE; n, number of cells.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
The closed times for 278283, W278G, Y279A, and R280G were longer than those for wild-type high-Po channels.
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
The data suggest that the histidine, arginine, and tryptophan residues interact with amiloride on the basis of the differences observed with the H282D/R, R280G/K, and W278G/A mutations and the level of disruption to amiloride binding caused by channels made from -subunits with these mutations.
The deletion of this region, 278283, and point mutations, W278G, R280G, F281A, and H282D, caused a change in the amiloride-blocking rate of the channel.
R280K
protein
substitution
true positive
P37089
Additional mutations, W278A, Y279F, and R280K, were generated via PCR using primers across the mutation site and subcloning the product back into a wild-type -rENaC/pCDNA3 backbone.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
These new mutations, W278A, Y279F, and R280K, were expressed in CHO cells and compared with wild-type -ENaC and the original mutations generated at these positions (Figs.
The restoration of amiloride sensitivity to wild-type levels by Y279F and R280K mutant channels supports the roles of these residues in the overall binding of amiloride to the wild-type channel.
AJP-Renal Physiol VOL WT R280G R280K W278G W278A Y279A Y279F 9 2 3 7 3 9 3 0.029 0.006 0.078 0.006 0.007 0.0004* 0.0004* 0.001* 1* 0.22 0.056* 0.03 0.001* Values are means SE; n, number of cells.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
W278A
protein
substitution
true positive
P37089
Additional mutations, W278A, Y279F, and R280K, were generated via PCR using primers across the mutation site and subcloning the product back into a wild-type -rENaC/pCDNA3 backbone.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
These new mutations, W278A, Y279F, and R280K, were expressed in CHO cells and compared with wild-type -ENaC and the original mutations generated at these positions (Figs.
AJP-Renal Physiol VOL WT R280G R280K W278G W278A Y279A Y279F 9 2 3 7 3 9 3 0.029 0.006 0.078 0.006 0.007 0.0004* 0.0004* 0.001* 1* 0.22 0.056* 0.03 0.001* Values are means SE; n, number of cells.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
F281A
protein
substitution
true positive
P37089
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
Two of the mutants, F281A and Y279A, which produced the most dramatic effects, caused a clear change in the way the channel transitioned from the open state to the closed state compared with wild-type channels.
The mean closed time was smaller for mutation F281A than for wildtype (low-Po) channels (Fig.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
ride include Y279A, F281A, and Y283A, with K0.5 values for amiloride of 0.22, 0.26, and 0.63 M, respectively (Table 4).
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
The deletion of this region, 278283, and point mutations, W278G, R280G, F281A, and H282D, caused a change in the amiloride-blocking rate of the channel.
H282R
protein
substitution
true positive
P37089
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
Previous work showed that deletion of this region as a whole ( 278283), as well as mutations of individual residues, R280G, H282D, and H282R, alters amiloride binding to the channel (12).
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
Y283A
protein
substitution
true positive
P37089
The mutant -subunits generated by these methods and used in expression were W278G, Y279A, R280G, F281A, H282D, H282R, and Y283A.
The Po for wild-type (high-Po) and all mutant channels are different, except for channels formed by the Y283A mutation (Fig.
Amiloride-Induced Changes in Po Are Altered in Mutant Channels Examination of the effect of amiloride on the Po of mutant channels addresses the role of the amino acids WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 6 9 3 9 3 10 3 2 3 9 10 3 5 0.851 0.128 0.012 0.14 0.16 0.064 0.045 0.030 0.018 0.18 0.43 0.25 0.39 0.030 0.495 0.043* 0.0193 0.004* 0.002 0.08* 0.016 0.08* 0.002 0.012* 0.010 0.017* 0.002 0.008* 0.215 0.011* 0.002 0.074* 0.008 0.13* 0.041 0.19* 0.140 0.22 0.018 0.215 0.006* 0.0003* 0.005* 0.0004* 0.002* 0.0002* 0.0001* 0.0001* 0.001* 0.025* 0.130* 0.007* 0.031 0.397 0.151 0.170 0.049 0.158 0.042 0.050 0.158 0.069 0.088 0.858 0.168 0.008 0.172 0.049 0.054 0.016 0.029 0.010 0.008 0.089 0.018 0.041 0.851 0.103 Values are means SE; n, number of Chinese hamster ovary (CHO) cells.
Po values are significantly lower for all mutant channels, except Y283A, than for wildtype (high-Po) channels (P 0.05).
ride include Y279A, F281A, and Y283A, with K0.5 values for amiloride of 0.22, 0.26, and 0.63 M, respectively (Table 4).
6 -ENaC Expressed in CHO Cells n K0.5, M WT 278283 W278G Y279A R280G F281A H282D H282R Y283A 9 5 7 9 2 4 4 3 4 0.029 0.006 0.22 0.006 0.26 0.022 0.63 0.007* 20* 0.001* 0.056* 0.0004* 0.082* 0.005* 1* 0.080* Values are means SE; n, number of cells.
Rate constants for closed, open, and blocked states of wild-type and mutant ENaC -ENaC Expressed in CHO Cells k1 , s 1 k 1, s 1 n k b, M 1 s 1 k b, s 1 n KAmil, M n WT High Po Low Po 278283 W278G W278A Y279A Y279F R280G R280K F281A H282D H282R Y283A 5.4 33 6.6 5.9 21 6.3 24 20 6.3 14 11 1.2 6.0 3.4 8.7 2.2 1.9 6.6 1.2 5.7 3.2 3.6 3.8 5.3 1.2 3.7 2.02 51.8 500 62.5 500 100 500 500 4.7 120 24 7.1 56 0.877 15.6* 75* 20* 100 20* 50 25* 2.3 16* 15 6.6 22 6 9 3 9 3 10 3 2 3 9 10 3 5 1,705 509 21 35 0.49 10 27 15 18 4 13 14 6 3 14 0.0247 0.25 0.00084 0.014 0.0014 0.00018 0.0014 0.047 0.0095 150 0.25 0.00602 0.23 0.0005* 3 0.004 0.00051 0.00005* 0.00018 0.024 0.004* 11* 0.46 15 5 7 3 9 3 2 3 4 4 3 4 141 50 592 296 ND 584 635 ND 6,209 5,428 ND 328 219 596 352 878 1,624 2,500 1,264 34 0.39 ND 8.2 9.3 ND 1.1 1.0 ND 15 13 5.6 3.9 1.3 105 610 1,200 Values are means SE; n, number of cells.
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
H2828R
protein
substitution
true negative
The point mutations appear to fall into two categories: those more sensitive to amiloride (W278G, R280G, and H282D) and those less sensitive to amiloride (Y279A, F281A, Y283A, and H2828R).
Typo
15044642
full text
W80C
protein
substitution
P51800
true positive
The new england journal of medicine 27.6 kb 4.3 kb fA AL355994 CLCNKA fB CLCNKB r 2.6 kb Ex3 Patient fA 23.2 kb fB CLCNKB Deletion of CLCNKB 1.7 kb r CLCNKA C C AG T G G C T G TA C Control fB r 4 kb 3 kb 2 kb fA r 4 kb 3 kb 2 kb C C A G T G C C T G T AC 4.3 kb 4.3 kb C C A G T G N C T GT A C Parent Patient Q79 W80C L81 Y82 Figure 2.
Effect of the ClC-Ka W80C Mutation on ClC-Ka Chloride Channel Activity.
Currents induced by wild-type ClC-Ka (Panel A) and ClCKa W80C (Panel B) in the presence of barttin are depicted.
The mean (SEM) of these five values (expressed as a percentage of the wild-type current) was 483 percent for ClC-Ka W80C.
A Wild-Type CIC-Ka+Barttin 3 2 Current (A) 1 0 1 2 3 200 400 600 800 Milliseconds B CIC-Ka W80C+Barttin 3 2 Current (A) 1 0 1 2 3 200 30 120 400 600 800 +40 0 gene, resulting in the substitution of cysteine (encoded by TGC) for tryptophan (encoded by the triplet codon TGG) at amino acid position 80 of the translated ClC-Ka protein (hereafter referred to as ClC-Ka W80C) (Fig.
Currents with identical biophysical properties but with severely reduced amplitudes (approximately half the amplitude of normal currents) were observed after coexpression of ClC-Ka W80C and barttin (Fig.
Milliseconds C 3 2 Wild-type CIC-Ka+barttin CIC-Ka W80C+barttin Wild-type CIC-Ka Current (A) 1 0 1 2 3 120 80 40 0 40 Voltage (mV) discussion In this report, we describe a digenic disorder involving impairment in the function of two chloride channels and resulting in a phenotype that combines severe renal salt wasting and deafness.
Indeed, the ClC-Ka W80C mutation identified in our patient severely impaired the function of his ClC-Ka chloride channels.
G240C
protein
substitution
true negative
The presence of a newly generated BsiYI site by the G240C mutation in exon 3 of the CLCNKA gene was ruled out by restriction analyses of PCR products derived from 102 control chromosomes from the 51 healthy control subjects.