Traffic Regulatory Proteins and ENaC

描述（由申请人提供）：钠通过肾和气道上皮面向管腔的膜进入是一个高度调控的过程，由上皮钠通道（ENaC）介导。ENaC功能异常与高血压、肾病、囊性纤维化和肺水肿等重大人类疾病有关。在肾上皮和气道上皮中，ENaC活性受控制顶端膜上活性通道数量的因子调节。细胞外液量和血压的关键激素调节因子响应的膜运输事件决定了顶端通道数。本研究旨在探讨肾上皮中ENaC转运的磷酸化依赖性调控。该领域几乎所有的研究都集中在ENaC从根尖膜恢复的机制上；相比之下，我们对ENaC向顶细胞表面的正向运输的调控机制知之甚少。在之前的资助期内，我们发现14-3-3蛋白是调节ENaC转运的磷酸化蛋白的重要稳定剂，并且我们开发了14-3-3亲和捕获作为识别ENaC正向转运重要调控节点蛋白的工具。因此，我们将研究这三种新的调节因子的作用机制，并评估它们的生理意义。首先，我们将定义lab - gap、AS160、14-3-3结合蛋白和醛固酮和胰岛素介导的ENaC转运的磷酸化依赖性调节因子的作用机制。相关蛋白TBC1D1是抗利尿激素刺激下根尖ENaC运输的候选调节因子，这些途径之间的相互作用可能解释了这些激动剂作用的协同作用。该方法还确定了肌动蛋白重组、14-3-3结合蛋白cofilin作为调控ENaC根尖插入的候选蛋白。我们的工作有望揭示根尖ENaC密度控制的新机制，并确定治疗钠转运上皮中异常盐和水平衡的新靶点。

DESCRIPTION (provided by applicant): The entry of sodium across the lumen-facing membranes of renal and airway epithelia is a highly regulated process, mediated by the epithelial sodium channel (ENaC). Abnormalities in ENaC function are implicated in significant human diseases, including hypertension, nephrosis, cystic fibrosis and pulmonary edema. In renal and airway epithelia, ENaC activity is regulated by factors that control the number of active channels residing in the apical membranes. Apical channel number is determined by membrane trafficking events in response to key hormonal regulators of the extracellular fluid volume and blood pressure. This proposal addresses the phosphorylation-dependent regulation of ENaC trafficking in renal epithelia. Nearly all research in this field has focused on the mechanisms that govern ENaC retrieval from the apical membrane; by contrast, our knowledge of the mechanisms that regulate the forward trafficking of ENaC to the apical cell surface is weak. During the prior funding period, we found that 14-3-3 proteins are essential stabilizers of the phospho-proteins that regulate ENaC trafficking, and we developed 14-3-3 affinity capture as a tool to identify proteins lying at important regulatory nodes in the forward trafficking of ENaC. Therefore, the proposed work will examine the mechanisms of action of three new regulators and assess their physiological significance. To begin, we will define the mechanism of action of the Rab-GAP, AS160, 14-3-3 binding protein and phosphorylation-dependent regulator of aldosterone- and insulin-mediated ENaC trafficking. A related protein, TBC1D1, is a candidate regulator of apical ENaC trafficking in response to vasopressin stimulation, and interactions between these pathways may account for synergism in the actions of these agonists. This approach has also identified the actin reorganizing, 14-3-3 binding protein, cofilin, as a candidate to control regulated apical ENaC insertion. Our work is expected to reveal new mechanisms for the control of apical ENaC density, and identify novel targets for the therapeutic targeting of abnormal salt and water balance in sodium transporting epithelia. PUBLIC HEALTH RELEVANCE: This proposal aims to identify the key regulators of epithelial sodium channel (ENaC) density at the apical membranes of renal epithelial cells. The trafficking of ENaC to the apical surface is the principal mode of channel regulation for hormones that sense extracellular fluid volume and blood pressure. These pathways are implicated in significant human diseases, including hypertension. Using 14-3-3 affinity methods, we have identified several new regulators of forward ENaC trafficking to the apical membranes, and we will define the mechanisms by which they control significant steps along the apical ENaC trafficking pathway in response to agonists. This work is expected to reveal new regulators and therapeutic targets for the control of sodium and fluid transport in the kidney.