Rokaw M D, Wang J M, Edinger R S, Weisz O A, Hui D, Middleton P, Shlyonsky V, Berdiev B K, Ismailov I, Eaton D C, Benos D J, Johnson J P
Laboratory of Epithelial Cell Biology, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
J Biol Chem. 1998 Oct 30;273(44):28746-51. doi: 10.1074/jbc.273.44.28746.
The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the beta subunit of xENaC in A6 cells. In vitro translated beta subunit, but not alpha or gamma, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when beta subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the beta subunit of xENaC.
醛固酮在反应性上皮细胞中增加顶端膜通透性的作用被认为是由于钠通道的激活。醛固酮刺激A6细胞顶端膜中一种95 kDa蛋白的甲基化，并且我们之前已经表明，免疫纯化的Na⁺通道复合物中95 kDa蛋白的甲基化增加了这些通道在平面脂质双分子层中的开放概率。我们在此报告，醛固酮刺激A6细胞中xENaC的β亚基的羧甲基化。体外翻译的β亚基，而非α或γ亚基，作为羧甲基化的底物。仅当存在β亚基时，重构于平面脂质双分子层中的ENaC的羧甲基化会导致开放概率增加。当从A6细胞中免疫沉淀通道复合物并用针对xENaC三个亚基的抗体进行蛋白质印迹分析时，所有三个亚基都被识别为复合物的组成成分。结果表明，A6细胞中的Na⁺通道活性部分受xENaC的β亚基的羧甲基化调节。
