Phosphorylation-dependent regulation of epithelial sodium channel (ENaC) traffick

描述（由申请人提供）：上皮钠通道（ENaC）在控制远端肾元钠重吸收中起着至关重要的作用。ENaC的调节对钠平衡和细胞外液容量的全面控制至关重要，从而对血压也至关重要。ENaC的失调是某些形式的原发性高血压的基础，原发性高血压是一种常见的疾病，也是心血管疾病和死亡的主要原因。然而，ENaC调控的分子机制尚不完全清楚。本研究旨在确定ENaC在肾上皮中转运的磷酸化依赖性调控。这一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，我们对促进ENaC向根尖细胞表面转运的机制的了解是不完整的。具体而言，尚未阐明顶膜ENaC循环和胞吐过程中磷酸化依赖的步骤和蛋白质相互作用。基于我们之前的发现，14-3-3蛋白是调节ENaC运输的磷酸化蛋白的重要稳定剂。14-3-3结合蛋白的亲和力捕获，结合定量蛋白质组学分析，为我们提供了极化肾上皮中ENaC交通调节的候选物。通过生化和功能分析，我们将评估影响ENaC根尖运输途径的14-3-3结合磷酸化蛋白。我们的初步数据表明，Rab-GAP蛋白AS160 （TBC1D4）和TBC1D1是响应醛固酮和抗利尿激素调节ENaC运输的蛋白激酶的关键底物。首先，我们将定义AS160的作用机制，AS160是一种新发现的14-3-3结合蛋白，是醛固酮介导的ENaC运输的磷酸化依赖调节剂。通过生化和功能分析，我们将验证AS160在基本条件下稳定ENaC在细胞内区室中的假设，并允许ENaC在其磷酸化的反应下运输到根尖膜。其次，我们将确定通过14-3-3亲和捕获确定的新靶点TBC1D1是否在抗利尿激素/PKA刺激下调节ENaC运输。这项工作有望揭示根尖ENaC密度控制的新机制，并为钠转运上皮中盐和水平衡疾病的治疗找到新的靶点。

DESCRIPTION (provided by applicant): The epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium reabsorption in the distal nephron. ENaC regulation is critical for the overall control of sodium balance and extracellular fluid volume, and thereby of blood pressure. Dysregulation of ENaC underlies some forms of essential hypertension - a common condition and a major cause of cardiovascular morbidity and mortality. However, the molecular mechanisms of ENaC regulation are not completely understood. This work aims to define 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 promote the regulated forward trafficking of ENaC to the apical cell surface is incomplete. Specifically, the phosphorylation-dependent steps and protein interactions involved in apical membrane ENaC recycling and exocytosis have not been elucidated. Based on our prior findings, 14-3-3 proteins are essential stabilizers of the phosphoproteins that regulate ENaC trafficking. Affinity capture of 14-3-3 binding proteins, combined with quantitative proteomic analysis, has given us candidates for the regulation of ENaC traffic in polarized renal epithelia. With biochemical and functional assays, we will evaluate the 14-3-3 binding phosphoproteins that impact the apical ENaC trafficking pathway. Our preliminary data has indicated that the Rab-GAP proteins, AS160 (TBC1D4) and TBC1D1, are key substrates for the protein kinases that regulate ENaC trafficking in response to aldosterone and vasopressin. To begin, we will define the mechanism of action of AS160, a newly identified 14-3-3 binding protein and phosphorylation-dependent regulator of aldosterone-mediated ENaC trafficking. Using biochemical and functional assays, we will test the hypothesis that AS160 stabilizes ENaC within intracellular compartments under basal conditions, and permits ENaC trafficking to the apical membrane in response to its phosphorylation. Second, we will determine whether TBC1D1, a new target identified by 14-3-3 affinity capture, regulates ENaC trafficking in response to vasopressin/PKA stimulation. This work is expected to reveal new mechanisms for the control of apical ENaC density, and identify novel targets for the treatment of diseases of salt and water balance in sodium transporting epithelia. PUBLIC HEALTH RELEVANCE: This study, titled "Phosphorylation-dependent regulation of epithelial sodium channel (ENaC) trafficking", will investigate whether phosphorylation-dependent 14-3-3 binding proteins stabilize multiple steps in the apical ENaC trafficking pathway, and whether their binding partners, AS160 (TBC1D4) and TBC1D1, are key substrates for the protein kinases that mediate the actions of aldosterone and vasopressin. Through this study, we will advance our understanding of molecular mechanisms of ENaC regulation and the results will provide further insight into the control of sodium balance, blood volume and thereby of blood pressure.