Molecular mechanism of voltage sensing in voltage-gated proton channels

Abstract

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels.

Figure 1.

Limiting slope estimates the effective gating charge. (A) Alignment of the S4 region (teal box) of Hv channels from C. intestinalis (Ci-Hv), Xenopus (XenHv), Mus musculus mouse (mHv), Homo sapiens (hHv), Gallus gallus (ChHv), and Danio rerio (DanHv) and S4 from Kv1.2, Shaker K, and domain IV of Nav1.4 channels. The arrow shows residues R1454 in Nav1.4 and R368 in Shaker that have been shown to move completely across the membrane during channel activation (Yang et al., 1996; Baker et al., 1998). Red letters indicate positively charged residues in S4. (B) Simulated conductance versus voltage, G(V), curve (black circles) for a three-state, two-subunit Hodgkin-Huxley type model with a gating charge of z = 2 e₀ per subunit. The G(V) was fit with a Boltzmann curve, G(V) = G0/(1 + exp(−z(V − V_1/2)/kT)) (z_fit = 2.43 e₀; dashed red line), and a Boltzmann curve to the second (z_fit = 2.0 e₀, resulting in a z_total(fit) = 2 × z_fit = 4.0 e₀; red line) and third power (z_fit = 1.87 e₀, resulting in a z_total(fit) = 3 × z_fit = 5.6 e₀; teal dashed line). (C) Simulated data and fits from B shown in lin-log plot (colored as in B). Also the best fit to the limiting slope at negative voltages is shown (log G(V) = constant + z_limitV/kT; z_limit(fit) = 3.8 e₀; teal straight line). (D) Currents from excised patches containing WT Ci-Hv1 channels (black line) or from patches taken from uninjected oocytes (teal line) in response to slow voltage ramps (1.5 mV/s) from −60 to 0 mV and then back to −60 mV. (E) The calculated conductance (I/(V − E_rev)) during the up and down ramp (data from D). Also shown is the calculated conductance from a patch from an uninjected oocyte (open circles). The currents were leak subtracted in response to ramps between −100 and −80 mV. The reversal potential E_rev was estimated to −85 mV in this experiment (pH_i = 5.5 and pH_o = 7). Note that the conductance estimated during the up ramp overlaps with the conductance during the down ramp, showing that the channels are at equilibrium at all voltages. This shows that the speed of the ramp is slow enough to correctly estimate the conductance. (F and G) Conductance, G, measured as in D from excised patches containing WT Ci-Hv1 channels in response to slow voltage ramps (1.5 mV/s) from −60 to 50 mV. The G(V) was fit with a Boltzmann curve, G(V) = G0/(1 + exp(−z(V − V_1/2)/kT)) (z_fit = 3.5 e₀; dashed red line), and a Boltzmann curve to the second power, G(V) = G0/(1 + exp(−z(V − V_1/2)/kT))² (z_fit = 3.0 e₀, resulting in a z_total(fit) = 2 × z_fit = 6.0 e₀; solid red line). (G) Data and fits from F shown in lin-log plot (colored as in F). Also the best fit to the limiting slope at negative voltages is shown (log G(V) = constant + z_limitV/kT; teal straight line). z_limit(fit) = 5.3 e₀.

Figure 2.

Neutralization of any S4 charge reduces the effective gating charge. (A) Currents from excised patches containing R255N Ci-Hv1 channels in response to slow voltage ramps (2 mV/s) from −60 to 40 mV and then back to −60 mV. (B) The current during the up and down ramp (data from A). (C–E) Conductance (G) calculated as G = 1/(V − E_rev) from measurements as in A and B from excised patches in response to slow voltage ramps (2 mV/s) for R255N (C), R258N (D), and R261N (E) Hv channels in pH_i = 5.5 and pH_o = 7. The limiting slope fitted as G = G₀/e^zδVF/RT is shown for each recording. (F) Effective gating charge (zδ) estimated from the limiting slope for charge-neutralized and WT Hv channels (n = 4) from experiments as in A and B. Error bars are SEM.

Figure 3.

Neutralization of S4 charges reduces the effective gating charge in monomeric Hv channels. (A) The current during up and down voltage ramps for monomeric R255N ΔNΔC Ci-Hv1 channels. (B) The calculated conductance (I/(V − E_rev)) during up and down voltage ramps for monomeric R258N ΔNΔC Ci-Hv1 channels. The conductance is similar during the up and down ramps, showing that the ramp speed is slow enough to estimate the steady-state conductance. (C) Effective gating charge (zδ) estimated from the limiting slope for charge-neutralized and WT Hv channels in dimeric (red) or monomeric (teal) form (ΔNΔC; see Materials and methods; n = 4). Error bars are SEM.

Figure 4.

R255N alters the extent of S4 movement. (A and B) Currents from an excised inside-out patch containing I262C channels in response to voltage steps from −40 to 80 mV, before (A) and after (B) application of internal MTSET. 1 mM MTSET was applied for 10 s on closed channels held at −100 mV in between the two recordings. (C and F) Currents from an excised inside-out patch containing R255N/I262C channels in response to voltage steps from −40 to 100 mV (in 20-mV increments), before (C) and after (F) 1 mM MTSET was applied 10 times at −100 mV for 1 s. pH_i = 5.5 and pH_o = 7. (D) Currents in response to a 100-mV voltage step every 10 s during MTSET application on open R2355N/I262C channels. The speed of the perfusion of the cytosolic face of the patch was tested by applying 1 mM MTSET (in pH 7.0 solution) at 100 mV for 1 s, followed by washout at 100 mV (pH 5.5 solution). Wash-in and washout were monitored by the fast changes in current amplitude induced by changes in internal pH: pH_i = 5.5 in rinse and pH_i = 7.0 in MTSET solution. pH_o = 7.0 (pipette solution). (E) Currents in response to a 100-mV voltage step every 10 s during MTSET application on closed R255N/I262C channels. Closed-state modification was tested by applying 1 mM MTSET at −60 mV for 1 s, followed by washout. Same patch as D. (G) Normalized currents in response to voltage steps to 100 mV after sequential 1-s applications of 1 mM MTSET applied at −100 mV on I262C (red boxes) and R255N/I262C (black boxes) channels, or sequential 1-s applications of 2 mM MTSET applied at −140 mV on R255N/I262C (black triangles) channels. MTSET was only applied during the inter-episode time at a holding voltage of −100 mV (or −140 mV; as in E). Error bars are SEM. (H and I) Cartoon showing proposed interactions of the positively charged S4 residues (R255, R258, and R261) with the two clusters of negatively charged residues in S1–S3 at the intracellular and extracellular borders of the Hv subunit in I262C (H) and R255N/I262C (I) channels. We propose that it is the interactions of the S4 charges with the two negatively charged clusters that set the limits for the extent of S4 movement (Fig. 3 [A and B] from Gonzalez et al., 2010).

Figure 5.

Cysteine accessibility is consistent with an S4 movement in which all three S4 charges significantly contribute to the effective gating charge. (A and B) Proposed S4 movement and charge–charge interactions in WT Hv channels according to present and previous (Gonzalez et al., 2010) cysteine accessibility data mapped onto the voltage-sensing domains from the Kv1.2-2.1 chimera channel structure (Long et al., 2007; A) and a closed-state model of Shaker K channels (Pathak et al., 2007; B). Positively charged S4 residues (R1, R2, and R3) and negatively charged glutamate residues (E1 and E2) are shown as ball and stick (except R2 = R258, which is space filled and colored yellow). Purple residues are only accessible from the extracellular solution in open channels and blue residues from the intracellular solution in closed channels (Gonzalez et al., 2010). Red residues (256 and 259) are not internally or externally accessible in closed or open channels (Gonzalez et al., 2010). Solid lines indicate proposed lipid bilayer boundaries, and dashed lines indicate proposed MTS accessibility as the result of water-filled crevices. Proposed positions of the charges of the three S4 arginine residues relative to the inaccessible portion of S4 are indicated. Assuming that the electrical field falls linearly over the inaccessible portion of S4, R255, R258, and R261 would contribute 0.80 e₀, 1.0 e₀, and 0.80 e₀, respectively, to a total gating charge Q = 2.6 e₀. This is similar to the contributions associated with these charges in FEP/MD simulations on the Kv1.2 voltage-sensing domain (Khalili-Araghi et al., 2010). (C and D) Two side views of the S1–S4 of an Hv1 channel monomer in the open (C) and closed (D) state rotated 180 degrees relative each other are shown in space fill. (E) Top view of the open state shown in C. (F) View from intracellular solution of the closed state shown in D. R258 is shown in yellow, and residues 256 and 259 are shown in red. R258 is accessible from the extracellular surface through a water-filled crevice (C and E) and from the intracellular surface through a water-filled crevice (D and F). Neither residue 256 nor 259 is visible from either side of the membrane in either closed or open states, consistent with earlier accessibility experiments (Gonzalez et al., 2010). Arrows show aqueous access to R258.

Figure 6.

Internal MTSET modifies R258C channels only in the closed state. (A and B) Currents from an excised inside-out patch containing 258C channels in response to voltage steps from −60 to 60 mV, before (A) and after (B) application of internal MTSET (1 mM) for 10 s and washout for 10 s. The holding potential was −60 mV. (C) Currents in response to a voltage step to 40 mV before and after application of internal MTSET. The current time course was fitted with an exponential (red lines) with τ = 70 ms before MTSET and 220 ms after MTSET. (D) Currents from an excised inside-out patch containing R258C channels in response to sequential voltage steps to 40, 100, and −100 mV. V_holding = −80 mV. pH_i = 5.5 and pH_o = 7. 1 mM MTSET was applied for 1 s during the −100-mV voltage step. This protocol was repeated every 10 s for 10 times (only four traces are shown for clarity). The inset shows currents during the 40-mV voltage step, showing the slowing of the current time course in response to consecutive MTSET applications. (E) The rate of MTSET modification at −100 mV (closed boxes) or 100 mV (open circles) was measured by fitting the current time course during the 40-mV voltage step (from data like in panel D) to a double exponential with τ1 = 70 ms (unmodified channels) and τ2 = 220 ms (modified channels). The amplitude of the slower component is plotted versus the cumulative MTSET exposure. The amplitude of the slower component is assumed to be proportional to the number of MTSET-modified 258C channels. The data were fitted with an exponential to yield the second order rate constant k for the MTSET reaction with R258C. k = 630/M/s for MTSET applied at −100 mV. No significant modification was seen when MTSET was applied only at 100 mV. n = 7. Error bars are SEM.

Figure 7.

External MTS reagents modify R258C channels only in the open state. (A and B) Currents from an oocyte expressing 258C channels in response to voltage steps from −50 to 50 mV, before (A) and after (B) application of external MTSEA (150 µM). The holding potential was −60 mV. MTSEA was applied at 40 mV for 65 s in between the two recordings, showing that 258C is accessible to the external solution in open channels. pH_o = 7.4. (C and D) Currents from an oocyte expressing 258C channels in response to voltage steps from −50 to 50 mV, before (C) and after (D) external application of membrane-impermeable MTSPT. The holding potential was −60 mV. 1 mM MTSPT was applied at 40 mV for 65 s in between the two recordings, showing that 258C is accessible to the external solution in open channels. pH_o = 7.4. (E) Sequential 5-s applications of 150 µM MTSEA followed by 10-s washout was applied at the indicated voltage, and then the current was monitored in response to a 40-mV step every 30 s. The rate of MTSEA modification at −80 mV (closed boxes) or 40 mV (open boxes) was measured using the time constant for the current activation (left y axis) for the first 1,000 ms after the start of the 40-mV voltage step. The activation time constant versus the cumulative MTSEA exposure was plotted and fitted to an exponential. As a control, 150 µM MTSEA was applied at 40 mV on WT Ci-Hv1 channels, and the time constant for activation was monitored (open triangles; right y axis). No change in the activation time constant was noticed for WT Ci-Hv1 channels. Error bars are SEM.