Structure-Function Studies of Epithelial Sodium Channel Gating

项目总结/摘要 上皮Na通道（ENaC）形成肾、肺和其他组织中Na+吸收的途径。 上皮细胞为了维持Na+稳态和控制血压，ENaC受到严格调节， 对Na+/容量耗尽和Na/容量过量的条件作出反应。然而，这种缺陷 调节是几乎所有已知的高血压遗传形式的原因，并有助于 囊性纤维化的发病机制。因此，我们的长期目标是了解 调节ENaC是开发这些疾病靶向治疗的先决条件。最近的一 发现的汇集将注意力集中在调节ENaC门控的机制上。在 在生物合成途径中，在细胞表面，蛋白酶切割ENaC和ENaC的胞外结构域， 将非活性通道转化为它们的活性Na+传导形式。此外，Na+调节ENaC门控 通过细胞外（Na+自我抑制）和细胞内（Na+反馈抑制）机制， 保持体内平衡其他细胞外分子也调节ENaC活性。但有 我们对这一现象背后的分子机制和通道结构的认识存在重大差距， 调控一个关键的进展是最近解决了一个密切相关的晶体结构 通道，ASIC 1.这提供了一个前所未有的结构，可能是基础的， 调节DEG/ENaC离子通道家族的门控。利用这些进步， 了解ENaC门控和ASIC 1晶体结构，本提案的总体目标是 了解调控ENaC门控的结构-功能关系。我们提出三个具体目标。 1.在初步研究中，我们发现细胞内Na+通过改变蛋白水解， ENaC和ENaC的分裂。在这个目标中，我们将测试Na+通过诱导一种新的蛋白质来改变卵裂的假设。 ENaC胞外结构域的构象变化。我们还将确定ENaC序列是 必需的. 2. ENaC暴露于肾脏和肺部的极端pH值。在初步研究中，我们发现 ENaC活性受细胞外pH调节。在这个目标中，我们将研究ENaC活性的分子机制。 的机制，并确定ENaC序列所需的pH值调节ENaC。3. ENaC是 也暴露于Cl-浓度的显著变化。我们的初步工作表明，氯- 调节ENaC电流，并为Na+自我抑制所需，这是细胞外Na+ 调节ENaC。因此，我们的目标是了解Cl-改变ENaC的机制 电流，并确定介导这种效果的胞外结构域中的残基。利用创新 方法和测试新的假设，这项工作将提供一个新的理解机制， 其调节ENaC门控，并因此调节上皮Na转运和Na稳态。

Project Summary/Abstract 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 ¿ and ¿ENaC, 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 ¿ and ¿ENaC. 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.