Structure-Function Studies of Epithelial Sodium Channel Gating

描述（由申请人提供）：上皮Na通道（ENaC）在肾、肺和其他上皮细胞中形成Na+吸收途径。为了维持Na+稳态和控制血压，ENaC受到严格调节，以应对Na+/容量耗尽和Na/容量过剩的情况。然而，这种调控的缺陷导致了几乎所有已知的遗传性高血压，并促进了囊性纤维化的发病机制。因此，我们的长期目标是了解调控ENaC的机制，作为开发针对这些疾病的靶向治疗的先决条件。最近的发现集中在调控ENaC门控的机制上。在生物合成途径和细胞表面，蛋白酶切割1和3ENaC的胞外结构域，将无活性通道转化为活性Na+传导形式。此外，Na+通过细胞外（Na+自抑制）和细胞内（Na+反馈抑制）机制调节ENaC门控以维持体内平衡。其他细胞外分子也调节ENaC的活性。然而，我们对这种调控背后的分子机制和通道结构的知识存在重大空白。一个关键的进展是最近解决了一个密切相关的通道ASIC1的晶体结构。这为DEG/ENaC离子通道家族的门控调节提供了一个前所未有的结构。利用对ENaC门控和ASIC1晶体结构的理解方面的这些进展，本提案的总体目标是了解调节ENaC门控的结构-功能关系。我们提出了三个具体目标。在初步研究中，我们发现细胞内Na+通过改变ENaC 1和3ENaC的蛋白水解裂解来调节ENaC。在这个目的中，我们将验证Na+通过诱导ENaC胞外结构域的构象变化来改变裂解的假设。我们还将确定所需的ENaC序列。2. ENaC暴露在肾脏和肺部的极端pH值下。在初步研究中，我们发现ENaC活性受细胞外pH调节。为此，我们将研究分子机制并确定pH调节ENaC所需的ENaC序列。3. ENaC也暴露于Cl-浓度的显著变化中。我们的初步研究表明Cl-调节ENaC电流，并且是Na+自抑制所必需的，这是细胞外Na+调节ENaC的机制。在这个目标中，我们的目标是了解Cl-改变ENaC电流的机制，并确定介导这种作用的细胞外结构域的残基。通过使用创新的方法和测试新的假设，这项工作将为ENaC门控的调节机制提供新的理解，从而为上皮Na转运和Na稳态提供新的理解。

DESCRIPTION (provided by applicant): The epithelial Na channel (ENaC) forms a pathway for Na+ absorption in the kidney, lung, and other epithelia. In order to maintain Na+ homeostasis and control blood pressure, ENaC is tightly regulated to respond to conditions of Na+/volume depletion and Na/volume excess. However, defects in this regulation are responsible for nearly all of the known inherited forms of hypertension, and contribute to the pathogenesis of cystic fibrosis. Thus, our long term objective is to understand the mechanisms that regulate ENaC as a prerequisite for the development of targeted treatments for these diseases. A recent convergence of discoveries has focused attention on mechanisms that regulate ENaC gating. In the biosynthetic pathway and at the cell surface, proteases cleave the extracellular domains of 1 and 3ENaC, converting inactive channels into their active Na+-conducting form. Moreover, Na+ regulates ENaC gating through extracellular (Na+ self-inhibition) and intracellular (Na+ feedback inhibition) mechanisms to maintain homeostasis. Other extracellular molecules also regulate ENaC activity. However, there are critical gaps in our knowledge about the molecular mechanisms and channel structures that underlie this regulation. A critical advance is the very recent solution of the crystal structure of a closely related channel, ASIC1. This has provided an unprecedented look at the structures that may underlie the regulation of gating of the DEG/ENaC ion channel family. Taking advantage of these advances in the understanding of ENaC gating and the ASIC1 crystal structure, the overall goal of this proposal is to understand structure-function relationships that regulate ENaC gating. We propose three Specific Aims. 1. In preliminary studies, we discovered that intracellular Na+ regulates ENaC by altering proteolytic cleavage of 1 and 3ENaC. In this aim, we will test the hypothesis that Na+ alters cleavage by inducing a conformational change in the ENaC extracellular domain. We will also identify the ENaC sequences are required. 2. ENaC is exposed to extremes of pH in the kidney and lung. In preliminary studies, we found that ENaC activity is regulated by extracellular pH. In this aim, we will investigate the molecular mechanisms and identify the ENaC sequences that are required for pH to regulate ENaC. 3. ENaC is also exposed to significant changes in Cl- concentration. Our preliminary work indicates that Cl- modulates ENaC current and is required for Na+ self-inhibition, a mechanism by which extracellular Na+ regulates ENaC. In this aim, our goal is to understand the mechanism(s) by which Cl- alters ENaC current, and to identify residues in the extracellular domains that mediate this effect. By using innovative approaches and by testing novel hypotheses, this work will provide a new understanding of mechanisms that regulate ENaC gating, and hence, epithelial Na transport and Na homeostasis. PUBLIC HEALTH RELEVANCE: Defects in the regulation of the epithelial sodium channel (ENaC) cause diseases including hypertension and cystic fibrosis. The overall goal of this proposal is to investigate the mechanisms that control the activity of ENaC. This will provide a new understanding of the pathogenesis of these diseases which will facilitate development of more effective treatments.