Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains.

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hH_V1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hH_V1, mutation of this motif did not affect cholesterol effects. The murine H_V1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the H_V1 protein. However, the data are fully explainable if H_V1 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_V1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hH_V1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hH_V1 binding to stomatin. The stomatin-mediated association of H_V1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct H_V1 to subcellular locations where it is needed, such as the phagosome in leukocytes.