Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19b, 14163, Berlin, Germany.
Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Oertzenweg 19b, 14163, Berlin, Germany.
Pflugers Arch. 2021 Dec;473(12):1859-1884. doi: 10.1007/s00424-021-02616-0. Epub 2021 Oct 19.
Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca signalling, but also for nitrogen metabolism. Models suggesting how influx of NH via TRPV3 might stimulate skin cornification or intestinal NH transport are discussed.
TRPV3 突变导致 Olmsted 综合征皮肤严重角化过度，但突变体是否能转运至细胞膜仍存在争议。尽管反刍动物和猪的研究表明 TRPV3 可能参与 NH 的摄取，但 TRPV3 在肠道上皮中的功能知之甚少。本研究旨在测量人同源物（hTRPV3）对 NH 的通透性，定位 hTRPV3 在人皮肤等效物中的位置，并研究 Olmsted 突变体 G573S 的转运。免疫印迹和免疫染色验证了 hTRPV3 在 HEK-293 细胞和非洲爪蟾卵母细胞中的成功表达，并转运至细胞膜。人皮肤等效物显示出顶层角质形成细胞的顶膜明显染色，中层有细胞质染色。非洲爪蟾卵母细胞 pH 敏感微电极实验表明，表达 hTRPV3 时，NH 酸化的程度显著增加。在过表达非洲爪蟾卵母细胞中的单通道测量显示出比对照更大的电导率。在 HEK-293 细胞的全细胞实验中，薄荷醇的两种对映异构体均刺激 hTRPV3 表达细胞中 NH 的内流，但在对照中没有。突变体 G573S 的表达大大降低了细胞活力，通过钌红部分挽救。免疫荧光证实了细胞质表达，在极少数细胞中观察到膜染色。我们认为上皮细胞表达 TRPV3 可能不仅对 Ca 信号转导有影响，而且对氮代谢也有影响。讨论了关于 NH 通过 TRPV3 内流如何刺激皮肤角化或肠道 NH 转运的模型。
