Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hH1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hH1, mutation of this motif did not affect cholesterol effects. The murine H1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the H1 protein. However, the data are fully explainable if H1 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 H1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hH1 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 hH1 binding to stomatin. The stomatin-mediated association of H1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct H1 to subcellular locations where it is needed, such as the phagosome in leukocytes.

中文翻译：

---

与造口素的相互作用将人类质子通道引导至胆固醇依赖性膜域

许多膜蛋白受胆固醇调节。在这里，我们报告了胆固醇消耗和恢复对切除斑块中的人类电压门控质子通道 hH1 的深远影响，但对全细胞结构的影响可以忽略不计。尽管存在假定的胆固醇结合位点（hH1 中的 CARC 基序），但该基序的突变并不影响胆固醇效应。鼠 H1 缺乏 CARC 序列，但表现出类似的胆固​​醇作用。这些结果反对胆固醇对 H1 蛋白的直接影响。然而，如果 H1 优先与胆固醇依赖性脂质结构域或“筏”结合，那么这些数据是完全可以解释的。筏预计会集中在膜/玻璃界面中，并因可电连接的贴片膜而耗尽。这一想法得到了证据的支持，即当施加吸力时，H1 通道可以在密封膜和补片膜之间扩散。同时截断 hH1 的大细胞内 N 和 C 末端大大减弱了胆固醇的作用，但单独截断 C 却没有；这表明 N 末端是脂质结构域的附着区域。寻找丰富的筏相关蛋白导致了stomatin。免疫共沉淀实验结果与hH1与stomatin的结合一致。气孔素介导的 H1 与胆固醇依赖性脂质结构域的关联为细胞提供了一种将 H1 引导至需要的亚细胞位置（例如白细胞中的吞噬体）的机制。