Structure-Function of the Epithelial Sodium Channel (ENaC)

描述(由申请人提供)：膜离子通道对于正确的电解质传输和液体平衡至关重要。上皮性钠通道(ENaC)位于电致密上皮细胞的管腔质膜上，如肾远端肾单位，对钠平衡的调节尤为重要。ENAC在维持液体和血压水平方面起着积极的作用，分别表现为功能丧失和功能获得遗传性疾病、利德尔综合征和假性低醛固酮I型。ENAC是一种成孔膜蛋白，由三个同源但不同的亚基组成。目前的建议旨在通过检查以下目标来提高我们对ENaC结构特征的理解：目标1：通过最近开发的ENaC功能的创新酵母筛选，将识别对保持ENaC结构和功能至关重要的残基和基序。将产生ENaC亚基内靶区的随机突变。在随后突变的ENaC亚基在酵母中表达后，我们将确定一组ENaC功能突变的丢失/获得，以供继续研究。这个潜在的关键残基的子集将被用来产生结构-功能关系的可信假说，这些假说将在哺乳动物表达系统中以更机械的方法使用经典的电生理学进行检验。目的2：结合作用的表面等离子体共振分析将确定ENaC亚基之间潜在的关键相互作用。首先将研究维持亚基间相互作用所必需的多肽片段的结合，然后是在特定目标1中确定的潜在相互作用区域。固定化环境对观察到的结合相互作用的影响将被确定。我们期望结合相互作用的量化将导致对关键的亚基间接触的更好的理解。

DESCRIPTION (provided by applicant): Membrane ion channels are critical for proper electrolyte transport and fluid balance. The epithelial sodium channel (ENaC) resident to the luminal plasma membrane of electrically tight epithelia, such as that lining the distal renal nephron, is, in particular, essential to regulation of sodium balance. ENaC plays an active role in the maintenance of fluid and blood pressure levels as seen with loss of function and gain of function genetic disorders, Liddle's syndrome and Pseudohypoaldosteronism type I, respectively. ENaC is a pore forming membrane protein composed of three homologous but distinct subunits. The current proposal is designed to improve our understanding of ENaC's structure features by examining the following aims: Aim 1: Residues and motifs that are critical for maintaining ENaC structure and function will be identified through an innovative yeast screen of ENaC function recently developed. Random mutation of targeted regions within ENaC subunits will be generated. After subsequent expression of mutated ENaC subunits in yeast we will identify a set of ENaC loss/gain in function mutations appropriate for continued studies. This sub-set of potential critical residues will be used to generate plausible hypotheses of structure-function relationships, which will be examined in a mammalian expression system using classical electrophysiology in a more mechanistic approach. Aim 2: Potentially critical interactions between ENaC subunits will be identified using surface plasmon resonance analysis of binding interactions. Binding of peptide fragments predicted to be essential for maintaining intersubunit interactions will be investigated first, followed by potential interacting regions identified in Specific Aim 1. The influence of immobilization environment on the observed binding interactions will be determined. We expect that quantification of binding interactions will lead to a greater understanding of critical intersubunit contacts.