During reverse cholesterol transport, high-density lipoprotein (HDL) carries excess cholesterol from peripheral cells to the liver for excretion in bile. The first and last steps of this pathway involve the HDL receptor, scavenger receptor BI (SR-BI). While the mechanism of SR-BI-mediated cholesterol transport has not yet been established, it has long been suspected that cholesterol traverses through a hydrophobic tunnel in SR-BI’s extracellular domain. Confirmation of a hydrophobic tunnel is hindered by the lack of a full-length SR-BI structure. Part of SR-BI’s structure has been resolved, encompassing residues 405 to 475, which includes the C-terminal transmembrane domain and its adjacent extracellular region. Within the extracellular segment is an amphipathic helix (residues 427–436, referred to as AH(427–436)) that showed increased protection from solvent in NMR-based studies. Homology models predict that hydrophobic residues in AH(427–436) line a core cavity in SR-BI’s extracellular region that may facilitate cholesterol transport. Therefore, we hypothesized that hydrophobic residues in AH(427–436) are required for HDL cholesterol transport. Here, we tested this hypothesis by mutating individual residues along AH(427–436) to a charged residue (aspartic acid), transiently transfecting COS-7 cells with plasmids encoding wild-type and mutant SR-BI, and performing functional analyses. We found that mutating hydrophobic, but not hydrophilic, residues in AH(427–436) impaired SR-BI bidirectional cholesterol transport. Mutating phenylalanine-430 was particularly detrimental to SR-BI’s functions, suggesting that this residue may facilitate important interactions for cholesterol delivery within the hydrophobic tunnel. Our results support the hypothesis that a hydrophobic tunnel within SR-BI mediates cholesterol transport.

在胆固醇逆向转运过程中，高密度脂蛋白 (HDL) 将多余的胆固醇从外周细胞运送到肝脏，以便在胆汁中排泄。该途径的第一步和最后一步涉及 HDL 受体、清道夫受体 BI (SR-BI)。虽然 SR-BI 介导的胆固醇转运机制尚未确定，但长期以来一直怀疑胆固醇穿过 SR-BI 细胞外结构域中的疏水隧道。缺乏全长 SR-BI 结构阻碍了疏水隧道的确认。SR-BI 的部分结构已被解析，包括残基 405 至 475，其中包括 C 端跨膜结构域及其相邻的细胞外区域。在细胞外区段内是一个两亲螺旋（残基 427-436，称为AH（427-436）），在基于NMR的研究中显示出对溶剂的增强保护。同源模型预测 AH(427–436) 中的疏水残基排列在 SR-BI 细胞外区域的核心腔中，这可能有助于胆固醇转运。因此，我们假设 AH（427-436）中的疏水残基是 HDL 胆固醇转运所必需的。在这里，我们通过将沿 AH(427-436) 的单个残基突变为带电残基（天冬氨酸）、用编码野生型和突变 SR-BI 的质粒瞬时转染 COS-7 细胞并进行功能分析来验证这一假设。我们发现 AH（427-436）中的疏水性而非亲水性残基突变会损害 SR-BI 双向胆固醇转运。苯丙氨酸 430 的突变对 SR-BI 的功能尤其有害，表明该残基可能促进疏水通道内胆固醇传递的重要相互作用。我们的结果支持这样的假设，即 SR-BI 内的疏水隧道介导胆固醇转运。