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转运和液体平衡至关重要。上皮钠通道（ENaC）驻留于电紧密上皮细胞的管腔质膜，例如衬在远端肾单位的管腔质膜，对于钠平衡的调节特别重要。ENaC在维持液体和血压水平方面起积极作用，如分别在功能丧失和功能获得遗传性疾病、Liddle综合征和I型假性醛固酮减少症中所见。ENaC是由三个同源但不同的亚基组成的成孔膜蛋白。目前的建议是为了提高我们对ENaC的结构特征的理解，通过检查以下目标：目标1：残基和基序，是维持ENaC的结构和功能的关键将通过一个创新的酵母筛选ENaC功能最近开发的确定。将产生ENaC亚基内靶向区域的随机突变。突变的ENaC亚基在酵母中的后续表达后，我们将鉴定一组适合于继续研究的ENaC功能突变的丧失/获得。该潜在关键残基的子集将用于产生结构-功能关系的合理假设，其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。目的2：ENaC亚基之间潜在的关键相互作用将被确定使用结合相互作用的表面等离子体共振分析。首先研究预测对维持亚基间相互作用至关重要的肽片段的结合，然后研究特异性目标1中确定的潜在相互作用区域。将确定固定化环境对观察到的结合相互作用的影响。我们预计，定量结合相互作用将导致更好地了解关键intersubunit接触。 公共卫生相关性：血压控制已成为延长生命的重要因素。因此，了解影响血压控制的因素对正确治疗至关重要。拟议的研究将提高我们对血压维持调节器之一的理解。

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. 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.