Carattino Marcelo D, Hughey Rebecca P, Kleyman Thomas R
Renal-Electrolyte Division, Department of Medicine, Pittsburgh, Pennsylvania 15261.
Renal-Electrolyte Division, Department of Medicine, Pittsburgh, Pennsylvania 15261; Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
J Biol Chem. 2008 Sep 12;283(37):25290-25295. doi: 10.1074/jbc.M803931200. Epub 2008 Jul 23.
Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the alpha subunit and at a single extracellular site within the gamma subunit. Channels lacking furin processing of the alpha subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK(186)) in the gamma subunit. We also demonstrated that the tract alpha D206-R231, between the two furin cleavage sites in the alpha subunit, as well as the tract gamma E144-K186, between the furin and prostasin cleavage sites in the gamma subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the alpha and gamma subunit inhibitory tracts, respectively. We examined whether release of either the alpha or gamma inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the alpha subunit (alphaR205A,R208A,R231Abetagamma) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of the alpha subunit, amiloride-sensitive currents in oocytes expressing channels carrying gamma subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (alphabetagammaR143A,DeltaE144-K186 and alphaR205A,R208A,R231AbetagammaR143A, DeltaE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the alpha and gamma subunit inhibitory tracts, alpha subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in the gamma subunit (alphabetagammaR143A,DeltaE144-K186) showed a significant reduction in the efficacy of block by the synthetic alpha-26 inhibitory peptide representing the tract alphaD206-R231. Our data indicate that removal of the inhibitory tract from the gamma subunit, in the absence of alpha subunit cleavage, results in nearly full activation of the channel.
上皮钠通道（ENaC）的成熟涉及在α亚基内的两个细胞外位点以及γ亚基内的单个细胞外位点进行弗林蛋白酶依赖性切割。缺乏α亚基弗林蛋白酶加工的通道活性非常低。我们最近在γ亚基中鉴定出一个依赖前列腺素的切割位点（RKRK(186)）。我们还证明，α亚基中两个弗林蛋白酶切割位点之间的α D206-R231片段，以及γ亚基中弗林蛋白酶和前列腺素切割位点之间的γ E144-K186片段，都是抑制域。弗林蛋白酶对ENaC的切割，随后是前列腺素的切割，分别由于α和γ亚基抑制性片段的释放，导致通道开放概率逐步增加。我们研究了α或γ抑制性片段的释放是否在激活通道中起主导作用。在非洲爪蟾卵母细胞中共表达前列腺素和野生型通道或缺乏α亚基弗林蛋白酶切割的突变通道（αR205A、R208A、R231Aβγ），导致全细胞电流增加到相似水平。以类似的方式且独立于α亚基的蛋白水解加工，在表达携带γ亚基的通道的卵母细胞中，amiloride敏感性电流在弗林蛋白酶切割位点发生突变且抑制性片段缺失（αβγR143A、ΔE144-K186和αR205A、R208A、R231AβγR143A、ΔE144-K186）时，显著高于表达野生型ENaC的卵母细胞。当通道缺乏α和γ亚基抑制性片段时，α亚基切割是通道完全激活所必需的。缺乏γ亚基弗林蛋白酶切割和抑制性片段的通道（αβγR143A、ΔE144-K186）对代表αD206-R231片段的合成α-26抑制肽的阻断效果显著降低。我们的数据表明，在没有α亚基切割的情况下，从γ亚基去除抑制性片段会导致通道几乎完全激活。
