Regulation of the human voltage-gated proton channel by membrane sterols

Cholesterol, a crucial component of eukaryotic cell membranes, plays a role in stabilizing the membrane and influencing the function of various membrane proteins, including ion channels. Research has previously demonstrated that cholesterol inhibits the human voltage-gated proton channel (hHv1) by modifying its S4 segment, the region responsible for sensing membrane voltage and regulating proton permeation.

In a new study, researchers explored the impact of cholesterol analogs and specific locations within the hHv1 channel that contribute to cholesterol inhibition. Utilizing site-directed mutagenesis and docking simulations, the team found that desmosterol, the precursor of cholesterol, significantly reduces cholesterol inhibition. To investigate the underlying mechanism, the researchers employed single-molecule Fluorescence Resonance Energy Transfer (smFRET) to observe changes in the channel's conformational states.

Their findings revealed that desmosterol mitigates cholesterol inhibition by promoting the intermediate and open states of the S4 segment. Furthermore, they identified several key residues in the hHv1 channel responsible for cholesterol inhibition, with Y141A in the S2 segment being particularly noteworthy. Mutating Y141 to alanine (Y141A) decreased cholesterol inhibition by approximately 3-fold.

The smFRET data suggested that the Y141A mutation facilitates the intermediate conformation of the S4 segment, thereby enhancing the attenuation of cholesterol inhibition.

The researchers also conducted docking simulations, which indicated that multiple residues across the transmembrane domain of the hHv1 channel, rather than being confined to a single pocket, are critical for cholesterol inhibition. These results not only pinpoint the molecular factor responsible for cholesterol inhibition in the hHv1 channel but also shed light on the connection between desmosterol and cholesterol conversion via the enzyme DHCR24 and pH homeostasis in various cells, such as phagocytes, cardiomyocytes, neurons, and microglial cells.