Elucidating the molecular mechanism of ENaC function

项目总结(来自原始申请) 上皮钠通道(ENaC)微调钠和水的平衡，是最主要的 通过远端肾单位钠的重吸收调节血压和容量。因此， ENAC是肾脏疾病和血压控制的关键治疗靶点。在这里我们 建议通过确定高分辨率来开发ENaC功能的分子模型 ENaC在不同功能状态下的结构。ENaC的分子研究已被证明是困难的 这在很大程度上是因为ENaCs是不稳定的多聚体完整膜蛋白，可被 位于胞外区的抑制域的蛋白分解；这些肽链是如何 发挥其影响仍未得到解决。我们的目标是开发表达和 未裂解构象和裂解构象中ENaC的纯化。我们将应用这些方法 为了阐明支持ENaC门控、离子渗透和 单粒子冷冻电子显微镜与电子显微镜相结合的变构调节 电生理学，以及明智地选择的生化和生物物理分析。这个 结构将提供对ENaC蛋白分解激活的分子基础的关键洞察 抑制域的位置和确定调节ENaC活性的关键区 为合理的、基于结构的治疗策略提供了第一份准确的蓝图。

Project summary (from original application) The epithelial sodium channel (ENaC) fine-tunes Na+ and water balance and is the primary regulator of blood pressure and volume through Na+ reabsorption in the distal nephron. Thus, ENaC is a critical therapeutic target for kidney diseases and blood pressure control. Here we propose to develop a molecular model for ENaC function by determining high-resolution structures of ENaC in different functional states. Molecular studies of ENaC have proven difficult largely because ENaCs are labile, multimeric integral membrane proteins that are activated by proteolysis of inhibitory domains located in the extracellular domain; how these peptidyl tracts exert their effects remains unresolved. Our goal is to develop methods for expression and purification of ENaC in the uncleaved and cleaved conformations. We will apply these methods to elucidate structure-based mechanisms underpinning ENaC gating, ion permeation, and allosteric modulation by combining single-particle cryo-electron microscopy and electrophysiology, together with judiciously chosen biochemical and biophysical assays. The structures will offer key insight into the molecular basis of proteolytic activation of ENaC providing positions of the inhibitory domains and identifying critical regions that mediate ENaC activity yielding the first accurate blueprints for rational, structure-based therapeutic strategies.