FS 1-Meena etal-CellReports 2022

Intestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, <em>Trpv6-/-</em> mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-Azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.肠上皮紧密连接破坏是酒精相关内毒素血症、全身炎症及多器官损伤的主要致病因素。乙醇与乙醛可通过升高细胞内钙离子（intracellular Ca²+）水平破坏紧密连接。本研究鉴定出瞬时受体电位阳离子通道亚家族V成员6（TRPV6）——一种钙离子选择性通透通道——为酒精诱导细胞内钙离子升高、肠屏障功能障碍及全身炎症的关键介导因子。乙醇与乙醛可在人结肠腺癌细胞（Caco-2）中诱导TRPV6离子电流。通过Caco-2细胞单层及小鼠肠道类器官开展的研究显示，TRPV6敲除或抑制可减轻乙醇与乙醛诱导的钙离子内流、紧密连接破坏及屏障功能障碍。此外，<em>Trpv6-/-</em>小鼠对酒精诱导的肠屏障功能障碍具有抵抗性。利用3-叠氮丁醇（3-Azibutanol）进行光亲和标记实验，鉴定出TRPV6中一个组氨酸为潜在的酒精结合位点。将该组氨酸与邻近的精氨酸进行替换，可降低乙醇激活的离子电流。本研究结果表明，TRPV6是酒精诱导肠屏障功能障碍与炎症的必需调控因子。能够下调TRPV6功能的分子，有望减轻酒精相关的组织损伤。