Electrophysiological recordings and a liposome-based assay showing the absence of chloride and proton conduction in TTYH proteins

The Tweety homologues (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1–3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows features reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.Tweety同源蛋白（Tweety homologues, TTYHs）是一类保守的真核膜蛋白家族成员，在大脑中分布丰富。此前的研究将三种人类旁系同源蛋白划归为可被钙离子或细胞肿胀激活的阴离子通道。为解析其尚未明确的三维结构及其与功能的关联，我们通过冷冻电子显微镜（cryo-electron microscopy）解析了人类TTYH1至TTYH3的三维结构。所有解析得到的结构均呈现出一致的二聚体膜蛋白特征：包含5个跨膜片段与一个延伸的胞外结构域。鉴于所有靶蛋白均未展现出与阴离子通道相符的结构特征，我们重新开展了功能实验验证，结果未检测到任何离子传导的相关迹象。与之相反，我们在从脂双层延伸出的胞外结构域内的一处延伸疏水口袋中观测到电子密度信号，这提示TTYH蛋白可能参与与膜内脂类化合物的相互作用。