The voltage sensor is responsible for ΔpH dependence in Hv1 channels

Abstract

The dissipation of acute acid loads by the voltage-gated proton channel (H_v1) relies on regulating the channel's open probability by the voltage and the ΔpH across the membrane (ΔpH = pH_ex - pH_in). Using monomeric Ciona-H_v1, we asked whether ΔpH-dependent gating is produced during the voltage sensor activation or permeation pathway opening. A leftward shift of the conductance-voltage (G-V) curve was produced at higher ΔpH values in the monomeric channel. Next, we measured the voltage sensor pH dependence in the absence of a functional permeation pathway by recording gating currents in the monomeric nonconducting D160N mutant. Increasing the ΔpH leftward shifted the gating charge-voltage (Q-V) curve, demonstrating that the ΔpH-dependent gating in H_v1 arises by modulating its voltage sensor. We fitted our data to a model that explicitly supposes the H_v1 voltage sensor free energy is a function of both the proton chemical and the electrical potential. The parameters obtained showed that around 60% of the free energy stored in the ΔpH is coupled to the H_v1 voltage sensor activation. Our results suggest that the molecular mechanism underlying the H_v1 ΔpH dependence is produced by protons, which alter the free-energy landscape around the voltage sensor domain. We propose that this alteration is produced by accessibility changes of the protons in the H_v1 voltage sensor during activation.

Keywords: Hv1; coupling; gating currents; pH dependence; voltage sensor.

Fig. 1.

Monomeric H_v1 currents were ΔpH dependent. Representative current recordings of monomeric H_v1 at (A) ΔpH = 0, (B) ΔpH = 1, and (C) ΔpH = 2. Insets show the tail currents in an expanded time and current scale. Note the superposition of tail currents at high voltages suggesting that the open probability reached its maximum value. (D) Mean G-V curves at the indicated pH_in/pH_ex obtained from the tail currents. Curves were fitted using a two-state Boltzmann distribution model (lines; Eq. 1 in text and SI Appendix, Table S1). (E) Mean activation time constant as a function of voltage at the indicated pH_in/pH_ex (Eq. 2 in text). Data are shown as mean ± SEM.

Fig. 2.

Gating currents of the monomeric D160N H_v1 mutant channel conserved their distinctive characteristics. Currents produced in patches of membranes expressing the monomeric D160N H_v1 mutant at symmetrical pH 7 are shown. A -P/8 subtraction protocol from a subholding potential of −90 mV was applied. (A) Superimposed traces of gating currents produced by depolarizations from a holding potential of −90 mV. Gating currents were elicited by voltages from −60 to 240 mV in 10 mV steps. (B) Superimposed traces of currents produced by a 200-mV depolarization pulse of increasing duration. Holding potential was −90 mV. (C) Superimposed traces of currents produced by an ON-recovery protocol consisting of two 200-mV depolarization pulses separated by returning to −90 mV at increasing durations. Holding potential was −90 mV. (D) A Cole–Moore shift effect was produced when a 200-mV depolarization was preceded by a prepulse at the indicated voltage.

Fig. 3.

Gating currents of monomeric D160N H_v1 mutant were ΔpH dependent. Currents produced in patches of membranes expressing the monomeric D160N H_v1 mutant at different pHs were measured. A -P/8 subtraction protocol from a subholding potential of −90 mV was applied. Representative recordings at (A) ΔpH = 0, (B) ΔpH = 1, and (C) ΔpH = 2 are shown. (D) Mean Q-V curves at the indicated pH_in/pH_ex. Curves were fitted using a two-state Boltzmann distribution model (lines; Eq. 4 and SI Appendix, Table S2). (E) Mean ON-gating current decay time constant as a function of voltage at the indicated pH_in/pH_ex. (Eq. 3 in text). Data are shown as mean ± SEM.

Fig. 4.

The Q-V and G-V curves were well fitted to the empirical equation accounting for the ΔpH-dependent shifts. The normalized charge or conductance was globally fitted using a least-squares minimization method to the empirical equation 1/{1 + exp[(ΔG₀ − 2.3εRTΔpH − zFV)/RT]} (Eq. 6). (A) Q-V curves of the monomeric D160N H_v1 mutant at different ΔpHs shown in Fig. 3D were globally fitted to Eq. 6. The lines are the curves obtained from the fit at the indicated ΔpH using the parameters ΔG₀ = 12.3 ± 0.3 kJ/mol, ε = 0.57 ± 0.02, and z = 0.91 ± 0.02. (B) G-V curves of the monomeric H_v1 at different ΔpHs shown in Fig. 1D were globally fitted to Eq. 6. The lines are the curves obtained from the fit at the indicated ΔpH using the parameters ΔG₀ = 8.9 ± 0.1 kJ/mol, ε = 0.51 ± 0.01, and z = 0.75 ± 0.01. V_0.5 and V₀ values calculated from the global fit parameters using Eq. 11 are listed in SI Appendix, Table S3.

Fig. 5.

Water densities of the H_v1 voltage sensor changed during activation. (A) The crystal structure of the intermediate-resting state of the mouse H_v1 (mH_v1-IR) was used as a template to build a homology model of the intermediate-resting state of the Ciona-H_v1 (CiH_v1-IR, red). The active model of CiH_v1 (CiH_v1-A, black) was built by displacing the S4 arginines in the alignments (above; R255, R258, and R261 in red). A representative snapshot of the 100 ns molecular dynamics simulation is shown below with the S4 arginines in licorice representation for the CiH_v1-IR (red) and CiH_v1-A (black). (B) The relative densities of the arginine residues and water with respect to bulk water (P/P₀) along the cylindrical pore axis (z-axis) for the CiH_v1 models. The CiH_v1-IR model maintains the arginines in a lower position (Left, red trace) compared to the CiH_v1-A model (Right, black trace). For both models, the water density is shown in the blue traces.