Renal-Electrolyte Division, Dept. of Medicine, S933 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261, USA.
Am J Physiol Renal Physiol. 2012 Jan 1;302(1):F1-8. doi: 10.1152/ajprenal.00330.2011. Epub 2011 Oct 12.
The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ∼70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ∼70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ∼70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.
上皮钠离子通道（ENaC）的激活机制独特，其三个同源亚基中的两个亚基的细胞外环中的抑制性片段通过蛋白水解切割而释放。虽然生物合成途径中的弗林蛋白酶（furin）切割从α-亚基释放一个抑制性片段并适度激活通道，但通过γ-亚基上的第二个抑制性片段的释放实现完全激活需要弗林蛋白酶的一次切割，然后在一个较远的位置被第二种蛋白酶，如组织蛋白酶 S（TMPRSS4）、组织蛋白酶原、纤溶酶或弹性蛋白酶，进一步切割。我们现在报告，在非洲爪蟾卵母细胞中与小鼠 ENaC 共表达的小鼠跨膜丝氨酸蛋白酶 4（TMPRSS4）与通道活性增加 2 到 3 倍以及γ-亚基独特的约 70kDa 羧基末端片段的产生相关，类似于我们之前观察到的与依赖于组织蛋白酶 S 的通道激活相关的约 70kDa γ-亚基片段。TMPRSS4 依赖性通道激活和产生约 70kDa 片段被依赖于组织蛋白酶 S 的切割位点（γRKRK186QQQQ）的突变部分阻断。当弗林蛋白酶和组织蛋白酶 S 切割位点之间的三个碱性残基（γK173Q、γK175Q 和 γR177Q）发生突变时，TMPRSS4 依赖性 ENaC 激活和 γ-亚基切割的完全抑制观察到，除了 γRKRK186QQQQ 以外。与弗林蛋白酶切割位点相关的四个碱性残基（γRKRR143QQQQ）的突变也阻止了 TMPRSS4 依赖性通道激活。我们得出结论，TMPRSS4 主要通过切割位于弗林蛋白酶切割位点远端的γK173-K186 片段内的碱性残基来激活 ENaC，从而从γ-亚基上释放先前定义的关键抑制性片段，该片段包含γR158-F168。
