Cholesterol affects voltage-gated proton channels indirectly.

Abstract

A number of membrane proteins, including ion channels, interact directly with cholesterol within the plasma membrane. This interaction may affect physiologically relevant properties of the protein. Often, protein-sterol interaction is facilitated by a cholesterol recognition amino acid consensus sequence (CRAC) domain with (L/V)-X1-5-(Y)-X1-5-(K/R) or the inverse sequence CARC (K/R)-X1-5-(Y/F)-X1-5-(L/V) (Fantini & Barrantes, 2013, Front. Physiol. 4:31). The human proton channel hHV1 contains a putative CARC sequence KNNYAAMV (amino-acid residues 131-138) within the extrafacial part of its S2 transmembrane domain. We explored effects of cholesterol depletion from or addition to the membrane. Modulation of cholesterol levels using methyl-β-cyclodextrin (MBCD) dramatically altered current amplitude in inside-out membrane patches. Mutations to the CARC domain did not alter the effects of cholesterol, indicating that cholesterol binding to the CARC domain is not responsible. We propose that hHV1 molecules preferentially associate with cholesterol-dependent domains within the membrane. Cholesterol might affect proton currents if varying cholesterol changes the distribution of hHV1 molecules in the membrane between the cholesterol-dependent lipid domains and the rest of the membrane. A previous study reported that suction increased proton currents in excised patches and this was interpreted as evidence for mechanosensitivity of the HV1 protein (Pathak et al, 2016, J. Gen. Physiol. 148:405). We have confirmed that suction applied to the pipette increases proton currents. This observation is consistent with our hypothesis since membrane tension will undoubtedly affect the association of individual Hv1 molecules with lipid domains without the need for bona fide channel mechanosensitivity. Increase in membrane tension would result in the increased proton currents due to the dispersal of condensed lipid domains in a similar way to the cholesterol depletion. This study provides the first indication that HV1 associates with membrane lipid domains, sometimes called lipid rafts.