Elucidating the molecular mechanism of ENaC function

项目摘要（来自原始申请） 上皮钠通道（ENaC）微调Na+和水平衡，是主要的 通过远端肾单位的Na+重吸收调节血压和容量。因此，在本发明中， ENaC是肾脏疾病和血压控制的关键治疗靶点。这里我们 建议通过确定高分辨率， 不同功能状态下的ENaC结构。ENaC的分子研究已经证明是困难的 这主要是因为ENaC是不稳定的多聚体整合膜蛋白， 位于细胞外结构域的抑制性结构域的蛋白水解;这些肽基束如何 其影响尚未得到解决。我们的目标是开发表达方法， 未裂解和裂解构象的ENaC的纯化。我们将应用这些方法 阐明ENaC门控、离子渗透和 通过结合单粒子冷冻电子显微镜和 电生理学，连同明智地选择的生物化学和生物物理测定。的 结构将提供关键的洞察ENaC蛋白水解激活的分子基础， 抑制结构域的位置和鉴定介导ENaC活性的关键区域 为合理的、基于结构的治疗策略提供了第一个准确的蓝图。

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.