A voltage-gated proton-selective channel lacking the pore domain

Abstract

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.

Figure 1. Biophysical properties of expressed H_v1 currents

Depolarizing voltage steps (5 mV increments) were applied to an H_v1-transfected HM1 cell (a–c) to elicit outward H⁺ currents and deactivating inward tail currents (−80 mV). TMA5.5_o, TMA6.5_o or TMA7.5_o (TMA6.5_i for all) were used to impose pH gradients indicated in the diagrams. a, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=0.1$, V_h = −40 mV (V_step = +30 mV to +80 mV, scale bar 0.2 nA, 1 s; b, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1$, V_h = −40 mV (V_step = −30 mV to +20 mV), scale bar 0.1 nA, 1 s; c, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$, V_h = −70 mV (V_step = −60 mV to −10 mV), scale bar 0.2 nA, 1 s. d, H_v1 currents at the end of the depolarizing step (I_step, open symbols) and the absolute value of I_tail (−80 mV, filled symbols) are plotted as a function of the step voltage. Data shown are from the cell shown in a–c. Circles, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=0.1$ (V_thr = 70 ± 5.8 mV, n = 3); triangles, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1.0$ (V_thr = 11.7 ± 2.1 mV, n = 6); squares, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$ (V_thr = −31.7 ± 3.3 mV, n = 3). e, Representative currents for E_rev measurement (V_h = −40 mV, V_step = +40 mV, V_tail = −100 mV to +40 mV). Small I_step (<200 pA) was chosen to minimize ${\mathrm{H}}_{\mathrm{i}}^{+}$ depletion (note constant outward current level). f, Monoexponential fits of I_tail were extrapolated to t = 0 and E_rev was estimated from the zero-current intercept of linear fits to the data. Open circles, TMA6.5_i, TMA5.5_o ( $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=0.1$); filled triangles, TMA6.5_i, TMA6.5_o ( $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1$); open squares, TMA6.5_i, TMA7.5_o ( $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$). Average E_rev values: E_rev = 53.3 ± 1.4 mV, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=0.1$, n = 10; E_rev = 0.9 ± 0.7 mV, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1.0$, n = 14; E_rev = −56.5 ± 2.6 mV, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$, n = 5. Data represent mean ± s.e.m. from n experiments. g, V_thr is plotted against the Nernst potential for H⁺ at 24 °C. The data are fitted to V_thr = 0.82 E_rev + 13.8 mV (solid line). Data represent mean ± s.e.m. from n = 3–7 experiments. The V_thr versus E_rev relationship for native G_vH⁺ (V_thr = 0.79 E_rev + 23 mV, dotted line) is shown for comparison.

Figure 2. H_v1 voltage-dependent gating

a, H_v1 currents (−60 mV to +120 mV, V_h = −40 mV, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$, Na6.5_i, Na7.5_o) in a representative cell. Scale bar 2 nA, 400 ms. Under symmetrical conditions (TMA6.5, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1$), τ_act = 715 ± 124 ms (+80 mV), n = 9 and τ_deact = 65.0 ± 12.1 ms (−80 mV), n = 7. b, R205A (−60 mV to +180 mV, V_h = −60 mV, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$, Na6.5_i, Na7.5_o). Scale bar 2 nA, 10 ms. Note the 40-fold difference in timescale compared to a. c, The I_tail–V relation (−80 mV, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=10$, Na6.5_i, Na7.5_o) was normalized to the maximum current obtained from a Boltzmann fit to the data for each cell expressing H_v1 (filled circles) or R205A (open circles) to estimate P_o − V. Data were fitted to a Boltzmann function (solid lines) and normalized to the extrapolated maximum current. Points represent mean ± s.e.m. of normalized data. A comparison of curve fits from individual experiments (H_v1, V_0.5 = 58.0 ± 5.6 mV, zδ = 0.90 ± 0.04, n = 6; R205A, V_0.5 = 99.5 ± 18.3 mV, zδ = 0.57* ± 0.03, n = 3; *P = 0.03 by Student’s non-paired t-test) indicates that significantly less effective charge is moved in R205A than in H_v1 during channel gating. Data represent mean ± s.e.m. from n experiments.

Figure 3. Mutations in H_v1 reveal residues required for Zn²⁺ inhibition

Inhibition of I_step (+25 mV to +50 mV) in cells superfused with EGTA-free TMA6.5 or Na6.5 ( $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1$) was typically faster than the sampling interval (10 s) and washout was always complete. Average IC₅₀ = 2.2 ± 0.6 μM, n_H = 1.0 ± 0.2, n = 4; $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1$, + 40 mV, TMA6.5. Data represent mean ± s.e.m. from n experiments. The applied voltages and [Zn²⁺] for the records shown (Na6.5, $\rho {\mathrm{H}}_{\mathrm{i}/\mathrm{o}}^{+}=1$) were: a, H_v1, V_step = +40/V_tail = −80 mV, [Zn²⁺] = 0, 0.1, 1, 10, 100 μM; b, H140A, +40/−60 mV, [Zn²⁺] = 0, 1, 10, 100 μM; c, H193A, +30/−80 mV, [Zn²⁺] = 0, 10, 100 μM; d, H140A/H193A, +50/−20 mV, [Zn²⁺] = 0, 1 mM. Scale bars: 100 pA, 1 s. e, Representative Zn²⁺ concentration-response curves for H_v1 (filled squares, IC₅₀ = 1.9 μM, n_H = 0.9), H193A (open triangles, IC₅₀ = 17.9 μM, n_H = 1.2), H140A (filled circles, IC₅₀ = 74.3 μM, n_H = 1.0), and H140A/H193A (open squares, maximum inhibition = 11.1 ± 3.4%, n = 3).