Mechanisms of ligand regulation in human voltage-gated proton channel.

Abstract

Voltage-gated proton (Hv) channels are standalone voltage sensors without separate pore-forming domains in canonical voltage-gated ion channels. They normally function as acid extruders for their unique voltage- and pH-dependent gating. Aside from voltage and pH, Hv channels are also gated by multiple cellular ligands, including Zn2+, polyunsaturated arachidonic acid, albumin, and membrane cholesterol, identified in our recent work. Our previous results have established the S4 segment as the central gating machinery in hHv1 channels by showing that voltage activation enriched its opening conformations while inhibitory ligands, including proton, Zn2+ and cholesterol, stabilized it at resting state conformations. How is the function of hHv1 channels regulated in cells? Our functional characterizations on purified hHv1 channels and those expressed in cells indicated that arachidonic acid has very strong activating effects on hHv1 channels, even being able to reverse both Zn2+ and cholesterol inhibition at physiological levels. Examining the structural effects of arachidonic acid on hHv1 channels using single-molecule FRET further revealed that it activates hHv1 channels by promoting ‘pre-opening’ rather than the opening conformations. In many cells, hHv1 channels are closely associated with NADPH oxidases to compensate for charge/pH imbalances, and both were found in cholesterol-rich lipid rafts. Our results propose that cholesterol perhaps determines the basal activities of the hHv1 channel in cells, while releases of arachidonic acid following irritation or injury may serve as a major mechanism to activate hHv1 channels to promote respiration burst mediating by NADPH oxidases.