Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 2012 Nov 30;287(49):40907-14. doi: 10.1074/jbc.M112.417519. Epub 2012 Oct 11.
A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.
越来越多的证据表明，上皮钠通道（ENaC）的细胞外结构域作为一种传感器，可根据细胞外环境的变化精细调节通道活性。我们之前发现酸性 pH 值会增加人 ENaC 的活性，这是由于 Na+ 自身抑制作用降低所致。在当前的工作中，我们确定了负责这种调节的细胞外结构域残基。我们发现，与人类 ENaC 相比，大鼠 ENaC 对 pH 值的敏感性较低，这种作用部分是由 γ 亚基介导的。我们在 γENaC 的细胞外结构域中鉴定出一组七个残基（Asp-164、Gln-165、Asp-166、Glu-292、Asp-335、His-439 和 Glu-455），当它们分别突变为 Ala 时，会降低 ENaC 对质子的激活作用。γ（E455）在 βENaC 中保守（Glu-446）；将该残基突变为中性氨基酸（Ala、Cys）会降低酸性 pH 值对 ENaC 的刺激作用，而用 MTSES 修饰 Cys 引入负电荷则会恢复 pH 值调节。将这七个 γENaC 突变与 β（E446A）组合产生的通道不能被酸性 pH 值激活，但碱性 pH 值的抑制作用仍然完整。此外，这些突变降低了 pH 值对 Na+ 自身抑制的影响。总之，这些数据确定了人 β-和 γENaC 中的八个细胞外结构域残基，它们是酸性 pH 值调节所必需的。
