Traffic Regulatory Proteins and ENaC

描述(由申请人提供)：钠通过肾和呼吸道上皮细胞面向管腔的膜进入是一个高度调节的过程，由上皮钠通道(ENaC)调节。ENaC功能异常与人类重大疾病有关，包括高血压、肾病、囊性纤维化和肺水肿。在肾和呼吸道上皮细胞中，ENaC的活性受控制存在于顶膜的活跃通道数量的因素的调节。心尖通道的数量是由细胞外液容量和血压的关键激素调节的膜转运事件决定的。这项建议解决了依赖于磷酸化的ENaC在肾上皮细胞运输的调节。几乎所有这一领域的研究都集中在控制ENaC从根尖细胞膜上恢复的机制上；相比之下，我们对ENaC正向运输到根尖细胞表面的机制的了解很弱。在之前的资助期间，我们发现14-3-3蛋白是调节ENaC运输的磷酸蛋白的必要稳定剂，我们开发了14-3-3亲和捕获作为一种工具来识别位于ENaC正向运输中重要调控节点的蛋白质。因此，这项拟议的工作将检查三个新的调节因子的作用机制，并评估它们的生理意义。首先，我们将确定RAB-GAP、AS160、14-3-3结合蛋白以及醛固酮和胰岛素介导的ENaC转运的磷酸化依赖调节因子的作用机制。相关蛋白TBC1D1是加压素刺激下顶端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，作为控制调节的顶端ENaC插入的候选。我们的工作有望揭示控制心尖部ENaC密度的新机制，并为钠转运上皮异常盐和水平衡的治疗靶点找到新的靶点。 公共卫生相关性：该提案旨在确定肾上皮细胞顶膜上皮钠通道(ENaC)密度的关键调节因素。对于感觉细胞外液容量和血压的激素来说，ENaC到根尖表面的运输是主要的通道调节方式。这些通路与包括高血压在内的重大人类疾病有关。利用14-3-3亲和方法，我们已经确定了几个新的ENaC正向转运到顶膜的调节因子，并将确定它们控制ENaC转运途径上的重要步骤以响应激动剂的机制。这项工作有望揭示控制肾脏内钠和液体运输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.