Structure and Function of Epithelial Sodium Channels

描述(由申请人提供)：上皮钠通道(ENaCs)是普遍存在的ENaC/DEG超家族的成员，这些超家族包括电压非依赖性、Na+选择性和阿米洛利敏感的三聚体离子通道。ENaCs在肾脏中高度表达，以异源三聚体的形式聚集，具有精致的Na+选择性孔道，这对微调Na+和K+平衡至关重要。从ENaC基因的转录到离子通道的运输和蛋白水解性激活，ENAC的功能受到多个水平的调控。ENaCs功能活动的异常调节对人类疾病，特别是影响全球约10亿人的高血压(高血压)起着重要作用。此外，尽管蛋白降解在ENaCs的激活中是不可或缺的，但ENaCs的异常蛋白降解导致了以肾病综合征为特征的氯化钠滞留。尽管ENaC具有如此重要的临床意义，但在原子分辨率下ENaC的作用机制仍不清楚，缺乏ENaC的X射线晶体结构一直是该领域取得进展的障碍。这项研究的目的是利用X射线结晶学、电生理学和其他生化和生物物理方法来解决ENaC组装、门控、离子渗透和变构调节的分子机制。目前，还没有大量生产用于生化和生物物理实验的异三聚体ENaCs的方法。这项研究应用的目的是开发一种技术来表达毫克量的ENaC，以证明适合于功能和结构分析的同质种群。这一应用的核心是确定ENaC在不同生理状态下的X射线晶体结构，这些研究将首次在原子水平上展示这些Na+选择性通道在静止、关闭和开放状态下的情况，从而有助于理解ENaC功能的分子基础。更重要的是，这些结构研究将成为未来治疗策略的蓝图。

DESCRIPTION (provided by applicant): Epithelial sodium channels (ENaCs) are members of the ubiquitous ENaC/DEG superfamily of trimeric voltage-independent, Na+-selective and amiloride-sensitive ion channels. Highly expressed in the kidneys, ENaCs assemble as heterotrimers that harbor an exquisitely Na+-selective pore that is critical in the fine tuning of Na+ and K+ balance. ENaC function is regulated at multiple levels from transcription of the ENaC genes, to the trafficking and the proteolytic activation of the ion channel. Abnormal regulation in the functional activity of ENaCs contributes importantly to human disease, and especially to hypertension (high blood pressure), a condition that affects about 1 billion people worldwide. Furthermore, although proteolysis is integral in the activation of ENaCs, aberrant proteolysis of ENaCs contributes to NaCl retention that characterizes nephrotic syndrome. Despite such clinical importance, detail into the mechanism of ENaC function at atomic resolution has remained elusive and the lack of x-ray crystal structures of ENaC has been a barrier to progress in the field. The objective of this research application is to resolve molecula mechanisms underlying ENaC assembly, gating, ion permeation, and allosteric modulation utilizing methods of x-ray crystallography, electrophysiology, and other biochemical and biophysical assays. At present, there are no methods for producing large quantities of heterotrimeric ENaCs for biochemical and biophysical experiments. The aim of this research application is to develop the technology to express milligram quantities of ENaC that demonstrate a homogenous population suitable for functional and structural assays. Central to this application is to determine the x-ray crystal structures of ENaC at different physiological states and these studies will provide the first atomic-level presentation of these Na+-selective channels in their resting, closed and open states, thus contributing to the understanding of the molecular basis of ENaC function. More importantly, these structural studies will serve as blueprints for future therapeutic strategies.