A segment of gamma ENaC mediates elastase activation of Na+ transport.
A segment of gamma ENaC mediates elastase activation of Na+ transport.
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DOI：
10.1085/jgp.200709781
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发表时间：
2007-12
期刊：
影响因子：
--
通讯作者：
Bridges RJ
中科院分区：
文献类型：
--
作者：
Adebamiro A;Cheng Y;Rao US;Danahay H;Bridges RJ
介导肾和肺中上皮细胞调节的Na+重吸收的上皮Na+通道（ENaC）可以被内源性蛋白酶（例如通道激活蛋白酶1）和外源性蛋白酶（例如胰蛋白酶和中性粒细胞弹性蛋白酶（NE））激活。外源蛋白酶激活通道的机制尚不清楚。为了检验ENaC上的残基介导蛋白酶依赖性通道激活的假设，使用三启动子人ENaC构建体在FRT上皮细胞系中稳定表达野生型和突变型ENaC，并在抑肽酶处理的细胞中测量蛋白酶诱导的短路电流激活。阿米洛利敏感性短路电流（INa）被醛固酮（1.5倍）和地塞米松（8倍）刺激。地塞米松处理的细胞用于所有后续研究。血清蛋白酶抑制剂抑肽酶使基线INa降低约50%，外源性添加胰蛋白酶、NE和猪胰弹性蛋白酶（PE）可使INa恢复至基线对照值，但凝血酶不能。因此，所有蛋白酶实验均在暴露于抑肽酶后进行。由于NE对底物的识别优先与活性位点的缬氨酸结合，因此α和γ ENaC细胞外环中的几个缬氨酸依次被甘氨酸取代。该扫描在γ ENaC的位置182和193处产生两个缬氨酸残基，当同时改变为其他氨基酸时，其导致对NE的抑制反应。突变导致激活速率降低和激活稳态电流水平降低。与在182和193位具有甘氨酸取代的突变体相比，NE对野生型ENaC的激活效率有20倍的差异。然而，突变体仍然容易被胰蛋白酶和相关的弹性蛋白酶PE激活。丙氨酸是PE的优选P1位置残基，并且γ亚基中丙氨酸190的取代消除了PE对INa的激活。此外，在γ亚基（γ Th）中的残基186处开始用新的凝血酶共有序列（LVPRG）取代允许凝血酶激活INa，而野生型ENaC无反应。MALDI-TOF质谱评估的23-mer肽的蛋白水解酶包括确定的残基（T176-S198）显示，水解发生在残基V193和M194之间的NE和A190和S191之间的PE。体外翻译研究表明，凝血酶切割γ Th，但不切割野生型γ亚基。这些结果表明，γ亚基缬氨酸182和193对于NE激活通道至关重要，丙氨酸190对于PE激活通道至关重要，并且可以通过插入新的凝血酶共有序列来实现通道激活。这些结果支持蛋白酶结合和γ亚基的可能切割导致ENaC活化的结论。
The epithelial Na+ channel (ENaC) that mediates regulated Na+ reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (INa) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline INa by approximately 50% and INa could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of α and γ ENaC were sequentially substituted with glycines. This scan yielded two valine residues in γ ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an ∼20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P1 position residue for PE and substitution of alanine 190 in the γ subunit eliminated INa activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the γ subunit (γTh) allowed for INa activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T176-S198) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the γTh but not the wild-type γ subunit. These results demonstrate that γ subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the γ subunit results in ENaC activation.
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影响因子：
3.8
作者：
Chra誰bi, A;Vallet, V;Firsov, D;Hess, SK;Horisberger, JD
通讯作者：
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DOI：
10.1073/pnas.0511184103
发表时间：
2006-02-21
影响因子：
11.1
作者：
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通讯作者：
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作者：
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DOI：
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发表时间：
2006-09-01
影响因子：
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作者：
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通讯作者：
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影响因子：
13.6
作者：
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通讯作者：
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