Structure-Function of the Epithelial Sodium Channel (ENaC)

描述（由申请人提供）：膜离子通道对于适当的电解质运输和流体平衡至关重要。上皮钠通道（ENaC）位于电紧上皮的管腔质膜上，例如位于远端肾元内的上皮钠通道，对钠平衡的调节尤为重要。ENaC在维持体液和血压水平方面发挥积极作用，这在功能遗传性疾病（Liddle综合征和I型假性低醛固酮增多症）的功能丧失和功能获得中分别可见。ENaC是一种孔隙形成膜蛋白，由三个同源但不同的亚基组成。目前的提案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来鉴定对维持ENaC结构和功能至关重要的残基和基序。ENaC亚基内的靶区会产生随机突变。在酵母中表达突变的ENaC亚基后，我们将在功能突变中确定一组ENaC损失/获得，适合继续研究。这一潜在关键残基子集将用于产生结构-功能关系的合理假设，这将在哺乳动物表达系统中使用经典电生理学以更机械的方法进行检查。目标2:ENaC亚基之间潜在的关键相互作用将通过结合相互作用的表面等离子体共振分析来确定。预计对维持亚基间相互作用至关重要的肽片段的结合将首先进行研究，然后是在Specific Aim 1中确定的潜在相互作用区域。将确定固定环境对观察到的结合相互作用的影响。我们期望结合相互作用的量化将导致对关键亚基间接触的更好理解。

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. PUBLIC HEALTH RELEVANCE: Blood pressure control has become an important factor in prolonging life. Therefore, an understanding of the factors that affect blood pressure control is critical to proper treatment. The proposed research will enhance our understanding of one of the regulators of blood pressure maintenance.