Human proton channels accumulate in cholesterol dependent membrane domains via direct interaction with stomatin

Many membrane proteins are modulated by cholesterol. Here we report strong effects of cholesterol depletion and restoration on the human voltage gated proton channel, hH _V 1 in excised patches but negligible effects in whole-cell configuration. Despite the presence of a putative cholesterol binding site, a CARC domain in the human voltage gated proton channel, hH _V 1, mutation of this domain did not affect cholesterol effects. The murine H _V 1 lacks a CARC sequence but displays similar cholesterol effects. These three results all argue against a direct effect of cholesterol on H _V 1. We propose that the data are explainable if H _V 1 preferentially associates with cholesterol-dependent lipid domains, or “rafts.” The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically-accessible patch membrane. This idea is supported by evidence that H _V 1 channels can diffuse between seal and patch membranes when suction is applied. Suction pulls membrane constituents including H _V 1 into the patch. In whole-cell studies moderate osmotic stretch does not noticeably alter H ⁺ currents. Simultaneous truncation of the large intracellular N- and C-termini greatly attenuated the cholesterol effect, but C-truncation only did not. We conclude that the N-terminus is the region of attachment to lipid domains. Searching for abundant raft-associated molecules led to stomatin. Co-immunoprecipitation experiments showed that hH _V 1 binds to stomatin. The stomatin-mediated association of H _V 1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct H _V 1 to subcellular location where it is needed, such as the phagosome in leukocytes.