Vacuolar H+ -ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells.
Vacuolar H+ -ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells.
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DOI：
10.1681/asn.2005121277
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发表时间：
2006-07
期刊：
影响因子：
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通讯作者：
Qiongqiong Yang;Guangmu Li;Satish K. Singh;E. Alexander;J. Schwartz
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文献类型：
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作者：
Qiongqiong Yang;Guangmu Li;Satish K. Singh;E. Alexander;J. Schwartz
空泡H+-ATPase B1亚基的点突变与远端肾单位分泌酸的能力受损(远端肾小管酸中毒)有关。为了验证这些突变干扰H+-ATPase组装和运输的假设，模拟遗传性远端肾小管酸中毒(M)或野生型(WT)B1已知点突变的构建体被导入大鼠内髓集合管细胞系，表达绿色荧光蛋白(GFP)-B1WT或GFP-B1M融合蛋白。在免疫共沉淀研究中，GFP-B1WT与其他H+-ATPase亚基(C、H和E)形成复合物，而GFP-B1M不形成。来自GFP-B1WT细胞的抗GFP抗体免疫沉淀的蛋白质具有ATPase活性，而来自GFP-B1M细胞的蛋白质不具有ATPase活性。质子泵介导的细胞内pH转运在GFP-B1M细胞中受到抑制，但在GFP-B1WT细胞中不受抑制。GFP-B1WT和GFP-B1M存在于细胞顶膜中，并随细胞酸化而增加。在GFP-B1WT细胞中，GFP-B1的顶膜部分、内源性B1和H+-ATPase的31-kD亚基随细胞酸化而增加。在GFP-B1M细胞中，内源性B1和31-kD亚基不随酸化而增加。B1点突变阻止了H+-ATPase的正常组装，也可能通过与内源性完整的H+-ATPase竞争运输到内髓集合管细胞而抑制H+-ATPase的功能。
Point mutations in the B1 subunit of vacuolar H+ -ATPase are associated with impaired ability of the distal nephron to secrete acid (distal renal tubular acidosis). For testing of the hypothesis that these mutations interfere with assembly and trafficking of the H+ -ATPase, constructs that mimic seven known point mutations in inherited distal renal tubular acidosis (M) or wild-type (WT) B1 were transfected into a rat inner medullary collecting duct cell line to express green fluorescence protein (GFP)-B1WT or GFP-B1M fusion proteins. In co-immunoprecipitation studies, GFP-B1WT formed complexes with other H+ -ATPase subunits (c, H, and E), whereas GFP-B1M did not. Proteins that were immunoprecipitated with anti-GFP antibody from GFP-B1WT cells had ATPase activity, whereas proteins from GFP-B1M cells did not. Proton pump-mediated intracellular pH transport was inhibited in GFP-B1M-transfected cells but not in GFP-B1WT cells. GFP-B1WT and GFP-B1M are present in the apical membrane and increased with cellular acidification. In GFP-B1WT cells, the apical membrane fraction of GFP-B1, endogenous B1, and the 31-kD subunits of the H+ -ATPase increased with cell acidification. In GFP-B1M cells, the endogenous B1 and 31-kD subunits did not increase with acidification. B1 point mutations prevent normal assembly of the H+ -ATPase and also may act as an inhibitor of H+ -ATPase function by competing with endogenous intact H+ -ATPase for trafficking in inner medullary collecting duct cells.