Electromechanical Coupling in Gating of the Hv1 Voltage Sensor is Revealed by Resting-State Currents in an S4 Arg to His Mutation (R205H)

Abstract

In the tetrameric Shaker voltage-gated K+ channel, the gating of a resting-state proton currents in the S4 mutant R362H reports a conformational change associated with voltage sensor (VS) activation (Starace, et al., 2004). Here we show that the analogous R205H mutation in the Hv1 proton channel mediates a novel H+ 'shuttle' conductance (GSH) that is gated off by membrane depolarization. The similarity of GSH in Hv1 R205H to the channel-like Shaker R362H suggests that GSH gating can be used to measure VS activation in Hv1. The intrinsic or ‘aqueous’ H+ conductance (GAQ) in Hv1 R205H occurs mainly at more positive voltages than GSH, producing a distinctive U-shaped G-V relation. The separation of GSH from GAQ gating along the voltage axis implies that initial voltage sensor activation precedes the opening of GAQ in Hv1. Changes in the transmembrane pH gradient that are known to shift the apparent voltage dependence of GAQ gating similarly affect GSH, indicating that pH-dependent gating occurs early in the Hv1 activation pathway. Second-site mutations of an S3 acidic residue (D185) that is selectively conserved in Hv1 orthologues shift the voltage dependence of GAQ gating, but do not alter GSH gating. D185 mutations are thus interpreted to perturb coupling between VS activation and the opening of GAQ. Another second-site mutation that results in voltage-dependent block of GAQ (R205H-N214R) does not affect the voltage dependence of either GSH or GAQ gating. The existence of VS-pore coupling in Hv1, which lacks a canonical pore domain, demonstrates that electromechanical coupling is not exclusive to tetrameric voltage-gated cation channels.