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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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The F463W and F463Y mutations dramatically slow channel activation. (A) Typical WT hERG currents recorded during an envelope of tails voltage protocol (inset). For clarity, capacity transients have been removed and current traces truncated such that only the tail currents are shown. The broken line represents the zero current level. To measure τact, peak tail current amplitudes were plotted against time and fit to a single exponential function. (B) Representative currents recorded from WT hERG channels during a deactivation protocol. Oocytes were held at −80 mV, depolarized to +60 mV for 500 ms to activate the channels, and then repolarized to potentials between −110 and +60 mV for 4 s. Tail currents were fit to a double exponential function and the value for τdeact was calculated as a weighted mean of the fast and slow time constants for the current decay. (C and D) Plot of τact (C) and τdeact (D) values for WT hERG and F463 mutant channels against the electrochemical potential for channel activation and deactivation, respectively (see Materials and methods). Because slower activating channels (e.g., F463W) were recorded using 15-s pulse durations, WT hERG data were plotted twice, using electrochemical potential energies calculated with ΔG0 values derived from G-V curves obtained using both 2- and 15-s pulse durations.
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fig3: The F463W and F463Y mutations dramatically slow channel activation. (A) Typical WT hERG currents recorded during an envelope of tails voltage protocol (inset). For clarity, capacity transients have been removed and current traces truncated such that only the tail currents are shown. The broken line represents the zero current level. To measure τact, peak tail current amplitudes were plotted against time and fit to a single exponential function. (B) Representative currents recorded from WT hERG channels during a deactivation protocol. Oocytes were held at −80 mV, depolarized to +60 mV for 500 ms to activate the channels, and then repolarized to potentials between −110 and +60 mV for 4 s. Tail currents were fit to a double exponential function and the value for τdeact was calculated as a weighted mean of the fast and slow time constants for the current decay. (C and D) Plot of τact (C) and τdeact (D) values for WT hERG and F463 mutant channels against the electrochemical potential for channel activation and deactivation, respectively (see Materials and methods). Because slower activating channels (e.g., F463W) were recorded using 15-s pulse durations, WT hERG data were plotted twice, using electrochemical potential energies calculated with ΔG0 values derived from G-V curves obtained using both 2- and 15-s pulse durations.

Mentions: To quantify deactivation kinetics, the decaying portion of tail currents (see Fig. 3 B) was fitted to a biexponential function of the form: I = A1e−t/τ1 + A2e−t/τ2 + C, where I is the current amplitude, t is time, A1 and A2 and τ1 and τ2 are the amplitudes and time constants for the slow and fast components, respectively, and C is a constant. For ease of comparison across multiple mutants, the weighted mean of the time constants was then calculated as: τdeact = (A1τ1 + A2τ2)/(A1 + A2).


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)

The F463W and F463Y mutations dramatically slow channel activation. (A) Typical WT hERG currents recorded during an envelope of tails voltage protocol (inset). For clarity, capacity transients have been removed and current traces truncated such that only the tail currents are shown. The broken line represents the zero current level. To measure τact, peak tail current amplitudes were plotted against time and fit to a single exponential function. (B) Representative currents recorded from WT hERG channels during a deactivation protocol. Oocytes were held at −80 mV, depolarized to +60 mV for 500 ms to activate the channels, and then repolarized to potentials between −110 and +60 mV for 4 s. Tail currents were fit to a double exponential function and the value for τdeact was calculated as a weighted mean of the fast and slow time constants for the current decay. (C and D) Plot of τact (C) and τdeact (D) values for WT hERG and F463 mutant channels against the electrochemical potential for channel activation and deactivation, respectively (see Materials and methods). Because slower activating channels (e.g., F463W) were recorded using 15-s pulse durations, WT hERG data were plotted twice, using electrochemical potential energies calculated with ΔG0 values derived from G-V curves obtained using both 2- and 15-s pulse durations.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753600&req=5

fig3: The F463W and F463Y mutations dramatically slow channel activation. (A) Typical WT hERG currents recorded during an envelope of tails voltage protocol (inset). For clarity, capacity transients have been removed and current traces truncated such that only the tail currents are shown. The broken line represents the zero current level. To measure τact, peak tail current amplitudes were plotted against time and fit to a single exponential function. (B) Representative currents recorded from WT hERG channels during a deactivation protocol. Oocytes were held at −80 mV, depolarized to +60 mV for 500 ms to activate the channels, and then repolarized to potentials between −110 and +60 mV for 4 s. Tail currents were fit to a double exponential function and the value for τdeact was calculated as a weighted mean of the fast and slow time constants for the current decay. (C and D) Plot of τact (C) and τdeact (D) values for WT hERG and F463 mutant channels against the electrochemical potential for channel activation and deactivation, respectively (see Materials and methods). Because slower activating channels (e.g., F463W) were recorded using 15-s pulse durations, WT hERG data were plotted twice, using electrochemical potential energies calculated with ΔG0 values derived from G-V curves obtained using both 2- and 15-s pulse durations.
Mentions: To quantify deactivation kinetics, the decaying portion of tail currents (see Fig. 3 B) was fitted to a biexponential function of the form: I = A1e−t/τ1 + A2e−t/τ2 + C, where I is the current amplitude, t is time, A1 and A2 and τ1 and τ2 are the amplitudes and time constants for the slow and fast components, respectively, and C is a constant. For ease of comparison across multiple mutants, the weighted mean of the time constants was then calculated as: τdeact = (A1τ1 + A2τ2)/(A1 + A2).

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