Cleavage in the γ-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels
Cleavage in the γ-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels
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DOI：
10.1113/jphysiol.2008.154435
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发表时间：
2008-10-01
影响因子：
5.5
通讯作者：
Korbmacher, Christoph
中科院分区：
文献类型：
--
作者：
Diakov, Alexei;Bera, Katarzyna;Korbmacher, Christoph
蛋白水解酶激活上皮钠通道(ENaC)的机制尚不完全清楚。我们研究了胞外蛋白水解酶对非洲爪哇卵母细胞异源表达大鼠ENaC的影响。应用胰酶使ENaC全卵母细胞电流增加约8倍，而不伴随通道表面表达的增加。胰酶的刺激作用在表达α-γ-ENaC的卵母细胞中被保留，但在表达α-β-ENaC的卵母细胞中被取消。因此，伽马亚基似乎是胞外蛋白水解酶激活通道所必需的。对γ亚基胞外环中一个可能的前列腺素裂解位点的定点突变表明，将181Lys残基突变为丙氨酸(Gamma K181a)增加了ENaC基线全卵母细胞电流，减少了通道表面的表达，并极大地降低了细胞外蛋白酶(胰蛋白酶、胰凝乳蛋白酶和人中性粒细胞弹性蛋白酶)的刺激作用。在从外向外斑块的单通道记录中，我们证明了Gamma K181a突变基本上取消了胰酶对接近静默的通道的激活，同时保留了胰蛋白酶对通道门控的刺激作用。使用β518C突变体ENaC的实验证实了胰酶对通道门控和对近静默通道的招募的这种明显的双重作用，该突变体可以通过暴露于巯基试剂以近1的开放概率转换为通道。有趣的是，Gamma K181a突变导致质膜上自发出现67 kDa的Gamma亚单位片段，这可以被Furin抑制剂阻止，也可以在细胞外胰酶激活通道后发生。这表明该突变促进了内源性蛋白水解酶对通道的切割和激活。这将降低近乎沉默的通道池，并解释突变通道的结构性激活和对细胞外蛋白水解酶的反应降低。我们得出结论，突变位点(K181a)影响ENaC的伽马亚基中的一个区域，该区域对于细胞外蛋白水解酶激活近沉默的通道具有重要的功能。
The mechanisms by which proteases activate the epithelial sodium channel (ENaC) are not yet fully understood. We investigated the effect of extracellular proteases on rat ENaC heterologously expressed in Xenopus laevis oocytes. Application of trypsin increased ENaC whole-oocyte currents by about 8-fold without a concomitant increase in channel surface expression. The stimulatory effect of trypsin was preserved in oocytes expressing alpha gamma-ENaC, but was abolished in oocytes expressing alpha beta-ENaC. Thus, the gamma-subunit appears to be essential for channel activation by extracellular proteases. Site-directed mutagenesis of a putative prostasin cleavage site in the extracellular loop of the gamma-subunit revealed that mutating the 181Lys residue to alanine (gamma K181A) increases ENaC baseline whole-oocyte currents, decreases channel surface expression, and largely reduces the stimulatory effect of extracellular proteases (trypsin, chymotrypsin and human neutrophil elastase). In single-channel recordings from outside-out patches we demonstrated that the gamma K181A mutation essentially abolishes the activation of near-silent channels by trypsin, while a stimulatory effect of trypsin on channel gating is preserved. This apparent dual effect of trypsin on channel gating and on the recruitment of near-silent channels was confirmed by experiments using the beta 518C mutant ENaC which can be converted to a channel with an open probability of nearly one by exposure to a sulfhydryl reagent. Interestingly, the gamma K181A mutation results in the spontaneous appearance of a 67 kDa fragment of the gamma-subunit in the plasma membrane which can be prevented by a furin inhibitor and also occurs after channel activation by extracellular trypsin. This suggests that the mutation promotes channel cleavage and activation by endogenous proteases. This would lower the pool of near-silent channels and explain the constitutive activation and reduced responsiveness of the mutant channel to extracellular proteases. We conclude that the mutated site (K181A) affects a region in the gamma-subunit of ENaC that is functionally important for the activation of near-silent channels by extracellular proteases.