Firsov D, Schild L, Gautschi I, Mérillat A M, Schneeberger E, Rossier B C
Institut de Pharmacologie et de Toxicologie, Université de Lausanne, Switzerland.
Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15370-5. doi: 10.1073/pnas.93.26.15370.
The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.
上皮性氨氯地平敏感钠通道（ENaC）控制着钠转运上皮细胞中的跨上皮钠运动，并且与利德尔综合征相关，利德尔综合征是盐敏感性高血压的一种常染色体显性形式。迄今为止，由于缺乏一种能够特异性和定量检测细胞表面表达的ENaC的方法，对ENaC通道特性以及导致利德尔综合征的突变的功能后果的详细分析受到了限制。我们基于针对在α、β和γ ENaC亚基各自的细胞外环中引入的FLAG报告表位的125I标记的M2抗FLAG单克隆抗体（M2Ab*）的结合开发了一种定量测定法。将FLAG表位插入ENaC序列中并未改变其功能和药理学特性。结合特异性和亲和力（Kd = 3 nM）使我们能够在单个非洲爪蟾卵母细胞中将宏观氨氯地平敏感钠电流（INa）与细胞表面表达的ENaC野生型和突变亚基的数量相关联。这些实验表明：（i）只有由α、β和γ ENaC亚基组成的异源多聚体通道在细胞表面最大程度且高效地表达；（ii）ENaC的总体开放概率比之前在单通道记录中观察到的低一个数量级；（iii）导致利德尔综合征的突变（βR564stop）通过两种机制增强通道活性，即通过增加ENaC细胞表面表达以及通过改变通道开放概率。这种定量方法为一种盐敏感性高血压形式的潜在分子机制提供了新的见解。
