Structural dynamics determine voltage and pH gating in human voltage-gated proton channel

Voltage-gated proton (Hv) channels are standalone voltage sensors without separate ion conductive pores. They are gated by both voltage and transmembrane proton gradient (i.e., ∆pH), serving as acid extruders in most cells. Like the canonical voltage sensors, Hv channels are a bundle of four helices (named S1 -S4), with the S4 segment carrying three positively charged Arg residues. Extensive structural and electrophysiological studies on voltage-gated ion channels, in general, agree on an outwards movement of the S4 segment upon activating voltage, but the real-time conformational transitions are still unattainable. With purified human voltage-gated proton (hHv1) channels reconstituted in liposomes, we have examined its conformational dynamics, including the S4 segment at different voltage and pHs using single-molecule fluorescence resonance energy transfer (smFRET). Here, we provide the first glimpse of real-time conformational trajectories of the hHv1 voltage sensor and show that both voltage and pH gradient shift the conformational dynamics of the S4 segment to control channel gating. Our results indicate that the S4 segment transits among three major conformational states and only the transitions between the inward and outward conformations are highly dependent on voltage and pH. Altogether, we propose a kinetic model that explains the mechanisms underlying voltage and pH gating in Hv channels, which may also serve as a general framework for understanding the voltage sensing and gating in other voltage-gated ion channels.