Traffic Regulatory Proteins and ENaC

描述（由申请方提供）：钠穿过肾脏和气道上皮细胞的腔面膜进入是一个高度调节的过程，由上皮钠通道（ENaC）介导。ENaC功能的缺失与重要的人类疾病有关，包括高血压、肾病、囊性纤维化和肺水肿。在肾和气道上皮中，ENaC活性受控制顶膜中活性通道数量的因子调节。顶端通道数量由响应于细胞外液体积和血压的关键激素调节剂的膜运输事件决定。该提案解决了肾上皮细胞中ENaC运输的磷酸化依赖性调节。几乎所有的研究在这一领域都集中在管理ENaC检索从顶端膜的机制，相比之下，我们的知识，调节ENaC的顶端细胞表面的正向贩运的机制是薄弱的。在之前的资助期间，我们发现14-3-3蛋白是调节ENaC运输的磷蛋白的基本稳定剂，并且我们开发了14-3-3亲和捕获作为鉴定位于ENaC正向运输中重要调控节点的蛋白质的工具。因此，拟议的工作将研究三种新调节剂的作用机制，并评估其生理意义。开始，我们将确定Rab-GAP，AS 160，14-3-3结合蛋白和醛固酮和胰岛素介导的ENaC运输的磷酸化依赖性调节剂的作用机制。一个相关的蛋白质，TBC 1D 1，是一个候选调节剂的顶端ENaC贩运在血管加压素刺激，这些途径之间的相互作用可能会导致协同作用，这些激动剂的行动。这种方法还确定了肌动蛋白重组，14-3-3结合蛋白，cofilin，作为一个候选人，以控制调节顶端ENaC插入。我们的工作有望揭示控制顶端ENaC密度的新机制，并确定钠转运上皮细胞中异常盐和水平衡的治疗靶点。 公共卫生相关性：该提案旨在确定肾上皮细胞顶端膜上皮钠通道（ENaC）密度的关键调节因子。ENaC向顶表面的运输是感知细胞外液体积和血压的激素的通道调节的主要模式。这些途径与包括高血压在内的重大人类疾病有关。使用14-3-3亲和方法，我们已经确定了几个新的监管机构的正向ENaC贩运顶端的膜，我们将定义的机制，通过它们控制重要的步骤沿着顶端ENaC贩运途径在响应激动剂。这项工作有望揭示控制肾脏钠和液体转运的新调节剂和治疗靶点。

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.