A Single S4 Arginine is Sufficient for Voltage Sensitivity in the Hv1 Proton Channel

Abstract

Voltage-gated proton conductances (GvH+) are found in a variety of cell types (e.g. alveolar epithelia and phagocytes) where they mediate an H+-selective transmembrane ion efflux that alkalinizes the cell and provides charge compensation for NADPH oxidase. The voltage sensor domain (VSD) protein Hv1 is required for native GvH+ and sufficient to reconstitute the hallmark biophysical features of GvH+ in heterologous expression systems. Conserved Arg residues in the S4 transmembrane helix of VSD proteins are believed to constitute the primary voltage sensing elements. Voltage-dependent conformational rearrangements of S4 thus drive channel gating. The Hv1 contains three putative voltage-sensing arginines (R205, R208 and R211) in S4. In order to examine the contribution of S4 Arg residues to voltage-dependent activation in Hv1, we mutated each to Ala and measured expressed H+ currents in voltage-clamped 293T cells. The effect of single mutations on the apparent threshold for voltage-dependent activation (VTHR) with symmetrical [H+] ranged from negligible (R205A) to +77 mV (R211A). In order to determine the minimal number of S4 arginines that are required for channel opening, we constructed double mutations in S4 (R205A-R208A, R208A-R211A and R205A-R208A). Mutations bearing a single Arg in either position 208 or 211 generated measurable H+ currents with dramatically shifted VTHR values (>+90 mV). A unique biophysical feature of GvH+ is the coupling of voltage and pH gradient sensing: voltage-dependent activation shifts ∼40mV per pH unit change in the H+ gradient (ΔpH = pHOUT - pHIN). Interestingly, the slope of the VTHR/ΔpH relation was similar to wt Hv1 for all of the mutations tested. Our results demonstrate that a single S4 Arg is sufficient for voltage and ΔpH sensing in Hv1 and suggest that S4 arginines differentially contribute to the voltage sensing mechanism.