Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 2011 Feb 25;286(8):6027-32. doi: 10.1074/jbc.M110.198127. Epub 2010 Dec 13.
The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of βENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αγβ orientation (listed clockwise when viewed from the top).
上皮钠通道 (ENaC) 的细胞外结构域暴露于肾脏中的广泛阴离子浓度中。我们之前已经证明细胞外 Cl(-) 抑制 ENaC 活性。为了确定参与 Cl(-) 抑制的位点，我们突变了 α、β 和 γENaC 细胞外结构域中与酸感应离子通道 1a 的 Cl(-) 结合位点同源的残基，并测试了 Cl(-) 对在非洲爪蟾卵母细胞中表达的 ENaC 活性的影响。我们在 ENaC 中鉴定出两个 Cl(-) 抑制位点。一个由 αENaC 的拇指结构域和 βENaC 的手掌结构域中的残基形成。该界面处的残基突变降低了 Cl(-) 抑制作用，并降低了 Na(+) 自身抑制作用。第二个位点由 βENaC 的拇指结构域和 γENaC 的手掌结构域之间界面的残基形成。这些残基的突变也降低了 Cl(-) 抑制作用，但对 Na(+) 自身抑制作用没有影响。相比之下，γENaC 的拇指结构域和 αENaC 的手掌结构域中的突变对 Cl(-) 抑制或 Na(+) 自身抑制作用几乎没有影响。数据表明，Cl(-) 通过两种不同的 Na(+)-依赖和 Na(+)-独立机制抑制 ENaC 活性，这两种机制对应于两个功能性 Cl(-) 抑制位点。此外，基于突变对 Cl(-) 抑制的影响、突变的加性性质以及 Cl(-) 抑制机制的差异，数据支持 ENaC 亚基以 αγβ 方向组装的模型（从顶部顺时针方向列出）。
