Cheng YM et al. (2013), Functional interactions of voltage sensor charg...

XB-ART-48970

J Gen Physiol 2013 Sep 01;1423:289-303. doi: 10.1085/jgp.201310992.

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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 .

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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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Species referenced: Xenopus
Genes referenced: gnao1 kcnh2 tbx2

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	Figure 1. Highly conserved amino acids in the S2 and S4 segments. Sequence alignment of the S2 region (left) and the S4 voltage sensor (right) of Shaker and hERG channels. Conserved residues forming the putative gating charge transfer center in S2 are highlighted in green. The positive S4 residues investigated in this study are highlighted in blue. The remaining S4 charges are shown in bold font. The conserved negative residues in S2 suggested to contribute to the gating charge transfer center in Shaker channels (i.e., E293) are shown in red.
	Figure 2. Bulky, aromatic substitutions of hERG F463 alter voltage-dependent gating. (A) Typical current traces recorded from WT and mutant hERG channels. Oocytes were held at −80 mV and subjected to either 2- or 15-s depolarizing steps to +60 V in 10-mV increments. Tail currents were recorded during a 2-s pulse to −60 mV. Arrows indicate the zero current level. Note the change in scale for F463W. (B) Effects of the F463 mutations on the G-V relationship. Peak tail currents from experiments such as those in A were normalized to the maximum peak tail current to provide a measure of conductance. WT data recorded using both 2- and 15-s pulses are shown to allow for comparisons with mutant G-V curves obtained using different pulse durations. Data points represent means ± SEM (error bars). Solid and broken lines indicate fits of the data to a Boltzmann function (see Materials and methods). n values and parameters from the Boltzmann fits are summarized in Table 1.
	Figure 3. 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.
	Figure 4. 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.
	Figure 5. The positive charge at position 538 is more important than that at 537 in controlling channel activation. (A–F) Typical current recordings from oocytes expressing various R537 and K538 single and double mutations. Arrows represent the zero current level. (A–C) Oocytes were held at −80 mV and subjected to either 2-s (A) or 10-s (B and C) pulses to +60 mV in 10-mV increments; tail currents were recorded at −60 mV. (D–F) Oocytes were held at −130 mV and subjected to 2-s pulses to 0 or 20 mV in 10-mV increments; tail currents were recorded at −130 mV. (G) Comparison of the effects of R537 mutations (R/K/Q) on the G-V relationship when a Lys residue is present at position 538. (H) Comparison of the effects of R537 (R/K/Q) mutations when the residue at position 538 is an Arg. (I) Comparison of the effects of R537 mutations (R/K/Q) on the G-V relationship when a Q residue is present at position 538. Lines in G–I represent fits of the data to a Boltzmann function. n values and Boltzmann parameters are summarized in Table 1. (J) Plot of the τact values against the electrochemical potential for activation for the R537 and K538 double mutants. Data points in G–J represent mean ± SEM (error bars).
	Figure 6. 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).
	Figure 7. K525 and K538 may interact with a conserved negative charge in S2. (A–D) Representative current traces from oocytes expressing the single D466E mutation and the double D466E/K525R, D466E/R537K, or D466E/K538R mutant constructs. Oocytes were held at −80 mV and subjected to either 2- or 10-s depolarizing pulses to +60 mV in 10-mV increments. Tail currents were recorded at −60 mV. Arrows indicate the zero current level. The exception to this was D466E/K525R, which was held at −110 mV, and tail currents were recorded at −130 mV. (E) Comparison of the G-V relationships for the D466E and D466E/K525R mutants. (F) Plot of the G-V relationships for the D466E and D466E/K525Q mutants. (G) Comparison of the G-V relationships for the D466E and D466E/R537K mutants. (H) Comparison of the G-V relationships for the D466E and D466E/K538R mutants. In E–H, lines represent fits of the data to a Boltzmann function. Data points represent mean ± SEM (error bars). n values and parameters from the Boltzmann fits are summarized in Table 1.
	Figure 8. Schematic representation of hERG channel gating. (A) Reaction coordinate diagram for hERG gating, based on the linear gating scheme proposed by Wang et al. (1997). Activation involves transitions through three closed (C) states to a single open (O) state; the inactivated state has been omitted for simplicity. The black line represents the gating reaction for WT channels; the red line highlights possible changes caused by the K525R mutation. These include a more negative value for ΔG0 consistent with stabilization of the O state and a decrease in the energy barrier (E(deact)) for the O → C2 transition to allow for faster deactivation. (B) The same reaction coordinate diagram in A is used to highlight the possible changes to the gating reaction caused by the K538R mutation (green line). These include a positive value for ΔG0 consistent with stabilization of the closed state and a large increase in the energy barriers for transitions between the C2 and O states that allows for the increases in the values of τact and τdeact. (C) Schematic showing the proposed functional interactions between the S4 gating charges and F463 and D466 in the S2 segment. Broken lines illustrate the interaction pairs thought to occur in the resting state (left) and the activated state (right).

References [+] :

Ahern, Focused electric field across the voltage sensor of potassium channels. 2005, Pubmed, Xenbase