Edinger Robert S, Yospin Jeremy, Perry Clint, Kleyman Thomas R, Johnson John P
Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2006 Apr 7;281(14):9110-7. doi: 10.1074/jbc.M509232200. Epub 2006 Feb 8.
Aldosterone acts to increase apical membrane permeability by activation of epithelial Na(+) channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the beta subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that beta mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na(+) transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na(+) transport induced by aldosterone at early time points (1-4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. beta ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.
醛固酮通过激活上皮钠通道（ENaC）来增加顶端膜通透性。我们之前已经表明，醛固酮在其作用过程早期通过刺激A6细胞中这种异源三聚体通道β亚基的甲基化来激活ENaC。醛固酮还刺激A6细胞中k-ras的表达和甲基化。为了确定醛固酮刺激的甲基化是否在哺乳动物细胞中出现，我们检测了醛固酮对源自小鼠皮质集合管（CCD）的连续培养上皮细胞系中甲基化和ras激活的影响，并确定β-mENaC是CCD细胞中一种酶进行甲基化的底物。醛固酮刺激CCD细胞中的蛋白质碱基不稳定甲基化。醛固酮在添加后1小时内刺激CCD细胞中的钠转运，并且在最初4小时内任何ENaC亚基的细胞量都没有增加。使用抑制剂3-脱氮腺苷抑制甲基化，在早期时间点（1 - 4小时）阻断了醛固酮诱导的钠转运刺激，而不影响任何ENaC亚基的细胞量。与抑制所有甲基化反应的3-脱氮腺苷（3-DZA）不同，小G蛋白甲基化或异戊二烯化的特异性抑制剂对早期醛固酮诱导的电流没有影响。甲基化ras的异戊烯基半胱氨酸羧甲基转移酶（PCMTase）的过表达对基础转运影响很小，但增强了醛固酮刺激的A6细胞转运。PCMTase在CCD细胞中的过表达对基础或醛固酮刺激的转运均无影响。此外，当在非洲爪蟾卵母细胞中共表达时，PCMTase对ENaC活性没有影响。醛固酮对CCD细胞中k-ras的信使RNA或蛋白质水平均无影响。通过搜索小鼠肾脏文库，我们鉴定出一种甲基转移酶，它能刺激非洲爪蟾卵母细胞中的ENaC活性，而不影响ENaC的表面表达。我们的结果表明，醛固酮刺激CCD细胞中的蛋白质甲基化，这是早期转运反应表达所必需的。在CCD细胞中，这种作用不是通过ras的甲基化介导的，因为醛固酮在这些细胞中不会诱导ras甲基化，并且甲基化ras的酶对ENaC活性没有直接影响。β-ENaC是CCD细胞中甲基化的底物。在CCD细胞中鉴定出一种直接刺激ENaC的新型甲基转移酶。
