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Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels.

Cheng YM, Hull CM, Niven CM, Qi J, Allard CR, Claydon TW - J. Gen. Physiol. (2013)

Bottom Line: As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state.However, hERG R537K did not stabilize the open state as predicted.Collectively, these data suggest a role for F463 in mediating closed-open equilibria, similar to that proposed for F290 in Shaker channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

ABSTRACT
Human ether-à-go-go-related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance-voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed-open equilibria, similar to that proposed for F290 in Shaker channels.

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Charge-conserving mutations in S4 modulate hERG voltage-dependent gating. (A) Typical current traces recorded from S4 mutant channels in response to the voltage protocols shown (insets). Note the change in scale for K538R. Arrows indicate the zero current level. (B) Comparison of the mean G-V relationships for WT hERG and the mutant channels shown in A. Lines represent fits of the data to a Boltzmann function. n values and Boltzmann parameters are summarized in Table 1. (C) Plot of τact values for WT hERG and the S4 mutant channels against the electrochemical potential for activation. The envelope of tails protocol described in Fig. 3 A was used to measure τact values over a range of voltages that depended on the V1/2 of the G-V curve for each mutant. (D) Comparison of τdeact values for WT hERG and the S4 mutant channels. τdeact was measured using the deactivation protocol described in Fig. 3 B, with a variable voltage range to accommodate the different shifts in the G-V curves caused by each mutant. For B–D, data points represent mean ± SEM (error bars). Similar to Fig. 3, electrochemical potential energies for WT hERG calculated using ΔG0 values derived from G-V curves obtained using both 2- and 10-s pulse durations are presented.
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fig4: Charge-conserving mutations in S4 modulate hERG voltage-dependent gating. (A) Typical current traces recorded from S4 mutant channels in response to the voltage protocols shown (insets). Note the change in scale for K538R. Arrows indicate the zero current level. (B) Comparison of the mean G-V relationships for WT hERG and the mutant channels shown in A. Lines represent fits of the data to a Boltzmann function. n values and Boltzmann parameters are summarized in Table 1. (C) Plot of τact values for WT hERG and the S4 mutant channels against the electrochemical potential for activation. The envelope of tails protocol described in Fig. 3 A was used to measure τact values over a range of voltages that depended on the V1/2 of the G-V curve for each mutant. (D) Comparison of τdeact values for WT hERG and the S4 mutant channels. τdeact was measured using the deactivation protocol described in Fig. 3 B, with a variable voltage range to accommodate the different shifts in the G-V curves caused by each mutant. For B–D, data points represent mean ± SEM (error bars). Similar to Fig. 3, electrochemical potential energies for WT hERG calculated using ΔG0 values derived from G-V curves obtained using both 2- and 10-s pulse durations are presented.

Mentions: We next examined the possibility that the reversal of the lysine and arginine residues at the extremes of the S4 segment (see Fig. 1) contributes to the differences in gating between hERG and Shaker channels. Thus, the effects of the charge conserving hERG K525R, and R537K mutations were assessed (Fig. 4). K525R had a left-shifted G-V relationship (Fig. 4 B) and a large, negative ΔΔG0 (Table 1), which is indicative of a destabilized closed state relative to the open state. This is consistent with previous findings in both hERG (Subbiah et al., 2004, 2005; Zhang et al., 2004, 2005) and Shaker channels (Tao et al., 2010) that a lysine residue at the top of the S4 segment stabilizes the closed state. At the bottom of S4, R537K had a right-shifted G-V relationship and a positive ΔΔG0, which is suggestive of a relative stabilization of the closed state. This is counter to what would be predicted from work in Shaker; the analogous Shaker K374 residue favors the open conformation of that channel more than a substituted arginine (i.e., K374R; Tao et al., 2010). Given this finding, the imperfect alignment between the hERG and Shaker sequences (Cheng and Claydon, 2012), and the related uncertainty surrounding the structure of the base of the hERG S4 segment, the K538R mutant was also characterized. Intriguingly, compared with R537K, the hERG K538R mutant caused an even greater right-shift of the G-V relationship and a more positive ΔΔG0 (Fig. 4 B and Table 1).


Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels.

Cheng YM, Hull CM, Niven CM, Qi J, Allard CR, Claydon TW - J. Gen. Physiol. (2013)

Charge-conserving mutations in S4 modulate hERG voltage-dependent gating. (A) Typical current traces recorded from S4 mutant channels in response to the voltage protocols shown (insets). Note the change in scale for K538R. Arrows indicate the zero current level. (B) Comparison of the mean G-V relationships for WT hERG and the mutant channels shown in A. Lines represent fits of the data to a Boltzmann function. n values and Boltzmann parameters are summarized in Table 1. (C) Plot of τact values for WT hERG and the S4 mutant channels against the electrochemical potential for activation. The envelope of tails protocol described in Fig. 3 A was used to measure τact values over a range of voltages that depended on the V1/2 of the G-V curve for each mutant. (D) Comparison of τdeact values for WT hERG and the S4 mutant channels. τdeact was measured using the deactivation protocol described in Fig. 3 B, with a variable voltage range to accommodate the different shifts in the G-V curves caused by each mutant. For B–D, data points represent mean ± SEM (error bars). Similar to Fig. 3, electrochemical potential energies for WT hERG calculated using ΔG0 values derived from G-V curves obtained using both 2- and 10-s pulse durations are presented.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: Charge-conserving mutations in S4 modulate hERG voltage-dependent gating. (A) Typical current traces recorded from S4 mutant channels in response to the voltage protocols shown (insets). Note the change in scale for K538R. Arrows indicate the zero current level. (B) Comparison of the mean G-V relationships for WT hERG and the mutant channels shown in A. Lines represent fits of the data to a Boltzmann function. n values and Boltzmann parameters are summarized in Table 1. (C) Plot of τact values for WT hERG and the S4 mutant channels against the electrochemical potential for activation. The envelope of tails protocol described in Fig. 3 A was used to measure τact values over a range of voltages that depended on the V1/2 of the G-V curve for each mutant. (D) Comparison of τdeact values for WT hERG and the S4 mutant channels. τdeact was measured using the deactivation protocol described in Fig. 3 B, with a variable voltage range to accommodate the different shifts in the G-V curves caused by each mutant. For B–D, data points represent mean ± SEM (error bars). Similar to Fig. 3, electrochemical potential energies for WT hERG calculated using ΔG0 values derived from G-V curves obtained using both 2- and 10-s pulse durations are presented.
Mentions: We next examined the possibility that the reversal of the lysine and arginine residues at the extremes of the S4 segment (see Fig. 1) contributes to the differences in gating between hERG and Shaker channels. Thus, the effects of the charge conserving hERG K525R, and R537K mutations were assessed (Fig. 4). K525R had a left-shifted G-V relationship (Fig. 4 B) and a large, negative ΔΔG0 (Table 1), which is indicative of a destabilized closed state relative to the open state. This is consistent with previous findings in both hERG (Subbiah et al., 2004, 2005; Zhang et al., 2004, 2005) and Shaker channels (Tao et al., 2010) that a lysine residue at the top of the S4 segment stabilizes the closed state. At the bottom of S4, R537K had a right-shifted G-V relationship and a positive ΔΔG0, which is suggestive of a relative stabilization of the closed state. This is counter to what would be predicted from work in Shaker; the analogous Shaker K374 residue favors the open conformation of that channel more than a substituted arginine (i.e., K374R; Tao et al., 2010). Given this finding, the imperfect alignment between the hERG and Shaker sequences (Cheng and Claydon, 2012), and the related uncertainty surrounding the structure of the base of the hERG S4 segment, the K538R mutant was also characterized. Intriguingly, compared with R537K, the hERG K538R mutant caused an even greater right-shift of the G-V relationship and a more positive ΔΔG0 (Fig. 4 B and Table 1).

Bottom Line: As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state.However, hERG R537K did not stabilize the open state as predicted.Collectively, these data suggest a role for F463 in mediating closed-open equilibria, similar to that proposed for F290 in Shaker channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

ABSTRACT
Human ether-à-go-go-related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance-voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed-open equilibria, similar to that proposed for F290 in Shaker channels.

Show MeSH
Related in: MedlinePlus