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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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F463 may interact with K525 and K538 to modulate activation gating. (A) Comparison of the mean G-V relationship for the F463W/K525R double mutation with those of the single mutants. (B) Comparison of the mean G-V relationships for the F463W/K538R double mutation with those of the single mutants. (C) Comparison of the G-V relationships for the F463W/R537K double mutation with those of the single mutants. Lines in A–C represent Boltzmann function descriptions of the data; n values and Boltzmann parameters are shown in Table 1. (D) Plot of the τact values against the electrochemical potential for activation for the channel constructs described in A and B. Data points represent mean ± SEM (error bars).
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fig6: F463 may interact with K525 and K538 to modulate activation gating. (A) Comparison of the mean G-V relationship for the F463W/K525R double mutation with those of the single mutants. (B) Comparison of the mean G-V relationships for the F463W/K538R double mutation with those of the single mutants. (C) Comparison of the G-V relationships for the F463W/R537K double mutation with those of the single mutants. Lines in A–C represent Boltzmann function descriptions of the data; n values and Boltzmann parameters are shown in Table 1. (D) Plot of the τact values against the electrochemical potential for activation for the channel constructs described in A and B. Data points represent mean ± SEM (error bars).

Mentions: We have shown that mutation of F463, as well as of K525 and K538, has strong effects on voltage-dependent gating of hERG channels. To determine whether interactions similar to those between F290 and S4 charges in Shaker channels also occur in hERG channels, the effects of combining the F463W and charge-conserving S4 mutations were examined. The G-V relationships of the F463W/K525R and F463W/K538R double mutants are displayed in Fig. 6 (A and B), alongside the relevant single mutations. Parameters for Boltzmann fits to the data are summarized in Table 1. Both double mutants had G-V relationships that were shallower and quite right-shifted relative to WT and the single F463W mutant channels. The ΔΔG0 values were large and positive (Table 1), which is consistent with a strong relative stabilization of the closed state. Also in agreement with closed state stabilization, the activation kinetics for the F463W/K525R and F463W/K538R were exceedingly slow and on par with those of the single F463W mutant (Fig. 6 D). Double mutant cycle analysis resulted in ΔΔG0,NA values well in excess of 4.2 kJ mol−1 for both F463W/K525R and F463W/K538R (Table 1), which strongly suggests that F463 is able to form functional interactions with both K525 and K538 during activation gating and is consistent with the possibility that F463 serves as a gating charge transfer center in hERG channels. In contrast, double mutant cycle analysis of the effects of the F463W/R537K double mutant on the G-V relationship (Fig. 6 C) resulted in a ΔΔG0,NA of 6.2 ± 1.1 kJ mol−1 (Table 1). Although this value suggests that R537 may be functionally coupled to F463, it is substantially lower than the ΔΔG0,NA for F463W/K538R, which is consistent with our earlier conclusion (Fig. 5 and Table 2) that K538 is likely more important than R537 in the regulation of steady-state activation.


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)

F463 may interact with K525 and K538 to modulate activation gating. (A) Comparison of the mean G-V relationship for the F463W/K525R double mutation with those of the single mutants. (B) Comparison of the mean G-V relationships for the F463W/K538R double mutation with those of the single mutants. (C) Comparison of the G-V relationships for the F463W/R537K double mutation with those of the single mutants. Lines in A–C represent Boltzmann function descriptions of the data; n values and Boltzmann parameters are shown in Table 1. (D) Plot of the τact values against the electrochemical potential for activation for the channel constructs described in A and B. Data points represent mean ± SEM (error bars).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig6: F463 may interact with K525 and K538 to modulate activation gating. (A) Comparison of the mean G-V relationship for the F463W/K525R double mutation with those of the single mutants. (B) Comparison of the mean G-V relationships for the F463W/K538R double mutation with those of the single mutants. (C) Comparison of the G-V relationships for the F463W/R537K double mutation with those of the single mutants. Lines in A–C represent Boltzmann function descriptions of the data; n values and Boltzmann parameters are shown in Table 1. (D) Plot of the τact values against the electrochemical potential for activation for the channel constructs described in A and B. Data points represent mean ± SEM (error bars).
Mentions: We have shown that mutation of F463, as well as of K525 and K538, has strong effects on voltage-dependent gating of hERG channels. To determine whether interactions similar to those between F290 and S4 charges in Shaker channels also occur in hERG channels, the effects of combining the F463W and charge-conserving S4 mutations were examined. The G-V relationships of the F463W/K525R and F463W/K538R double mutants are displayed in Fig. 6 (A and B), alongside the relevant single mutations. Parameters for Boltzmann fits to the data are summarized in Table 1. Both double mutants had G-V relationships that were shallower and quite right-shifted relative to WT and the single F463W mutant channels. The ΔΔG0 values were large and positive (Table 1), which is consistent with a strong relative stabilization of the closed state. Also in agreement with closed state stabilization, the activation kinetics for the F463W/K525R and F463W/K538R were exceedingly slow and on par with those of the single F463W mutant (Fig. 6 D). Double mutant cycle analysis resulted in ΔΔG0,NA values well in excess of 4.2 kJ mol−1 for both F463W/K525R and F463W/K538R (Table 1), which strongly suggests that F463 is able to form functional interactions with both K525 and K538 during activation gating and is consistent with the possibility that F463 serves as a gating charge transfer center in hERG channels. In contrast, double mutant cycle analysis of the effects of the F463W/R537K double mutant on the G-V relationship (Fig. 6 C) resulted in a ΔΔG0,NA of 6.2 ± 1.1 kJ mol−1 (Table 1). Although this value suggests that R537 may be functionally coupled to F463, it is substantially lower than the ΔΔG0,NA for F463W/K538R, which is consistent with our earlier conclusion (Fig. 5 and Table 2) that K538 is likely more important than R537 in the regulation of steady-state activation.

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