Voltage-gated ion channels are key players of electrical signaling in cells. As a unique subfamily, voltage-gated proton (Hv) channels are standalone voltage sensors without separate ion conductive pores. Hv channels are gated by both voltage and transmembrane proton gradient (i.e ∆pH), serving as acid extruders in most cells. Amongst their many functions, Hv channels are known for regulating the intracellular pH of human spermatozoa and compensating for the charge and pH imbalances caused by NADPH oxidases in phagocytes. Like the canonical voltage sensors, Hv channels are a bundle of 4 helices (named S1 through S4), with the S4 segment carrying 3 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 3 major conformational states and kinetic analysis suggest that only the transitions between the inward and outward conformations are highly dependent on voltage and pH changes. Our smFRET studies uncover the stochastic conformational dynamics of S4 and demonstrate how voltage and pH shift its conformational distributions to regulate channel gating. 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.

Although human sperm is morphologically mature in the epididymis, it cannot fertilize eggs before capacitation. Cholesterol efflux from the sperm plasma membrane is a key molecular event essential for cytoplasmic alkalinization and hyperactivation, but the underlying mechanism remains unclear. The human voltage-gated proton (hHv1) channel functions as an acid extruder to regulate intracellular pHs of many cell types, including sperm. Aside from voltage and pH, Hv channels are also regulated by distinct ligands, such as Zn 2+ and albumin. In the present work, we identified cholesterol as an inhibitory ligand of the hHv1 channel and further investigated the underlying mechanism using the single-molecule fluorescence resonance energy transfer (smFRET) approach. Our results indicated that cholesterol inhibits the hHv1 channel by stabilizing the voltage-sensing S4 segment at resting conformations, a similar mechanism also utilized by Zn 2+ . Our results suggested that the S4 segment is the central gating machinery in the hHv1 channel, on which voltage and distinct ligands are converged to regulate channel function. Identification of membrane cholesterol as an inhibitory ligand provides a mechanism by which the hHv1 channel regulates fertilization by linking the cholesterol efflux with cytoplasmic alkalinization, a change that triggers calcium influx through the CatSper channel. These events finally lead to hyperactivation, a remarkable change in the mobility pattern indicating fertilization competence of human sperm.

Voltage-gated ion channels are key players of electrical signaling in cells. As a unique subfamily, 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 ΔpH), serving as acid extruders in most cells. Amongst their many functions, Hv channels are known to regulate the intracellular pH of human spermatozoa and compensate for the charge and pH imbalances caused by NADPH oxidases in phagocytes. Like the canonical voltage sensors, the Hv channel is a bundle of 4 helices (named S1 through S4), with the S4 segment carrying 3 positively charged Arg residues. Extensive structural and electrophysiological studies on voltage-gated ion channels generally 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) channel reconstituted in liposomes, we have examined its conformational dynamics at different voltage and pHs using the single molecule fluorescence resonance energy transfer (smFRET). Here we provided the first glimpse of real time conformational trajectories of the hHv1 voltage sensor and showed that both voltage and pH gradient shift the conformational dynamics of the S4 segment to control channel gating. Our results suggested the biological gating is determined by the conformational distributions of the hHv1 voltage sensor, rather than the conformational transitions between the presumptive ‘resting’ and ‘activated’ conformations. We further identified H140 as the key residue sensing extracellular pH and showed that both the intracellular and extracellular pH sensors act on the voltage sensing S4 segment to enrich the resting conformations. Taken together, we proposed a model that explains the mechanisms underlying voltage and pH gating in Hv channels, which may also serve as a general framework to understand the voltage sensing and gating in other voltage-gated ion channels.

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