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

描述（由申请人提供）：上皮钠通道（ENaC）在远端肾单位钠重吸收的控制中具有根本重要性。ENaC调节对于钠平衡和细胞外液量的总体控制以及血压的总体控制至关重要。ENaC的失调是某些形式的原发性高血压的基础，这是一种常见的疾病，也是心血管疾病发病率和死亡率的主要原因。然而，ENaC调控的分子机制尚未完全了解。 这项工作的目的是确定磷酸化依赖的调控ENaC运输在肾上皮细胞。几乎所有的研究在这一领域都集中在管理ENaC检索从顶膜的机制。相比之下，我们的知识的机制，促进调节前向贩运ENaC顶端细胞表面是不完整的。具体而言，磷酸化依赖的步骤和蛋白质相互作用参与顶端膜ENaC再循环和胞吐尚未阐明。 根据我们之前的发现，14-3-3蛋白是调节ENaC运输的磷蛋白的重要稳定剂。亲和捕获14-3-3结合蛋白，结合定量蛋白质组学分析，为我们提供了在极化的肾上皮细胞中调节ENaC交通的候选人。通过生物化学和功能测定，我们将评估影响顶端ENaC运输途径的14-3-3结合磷蛋白。我们的初步数据表明，Rab-GAP蛋白，AS 160（TBC 1D 4）和TBC 1D 1，是调节ENaC运输的蛋白激酶的关键底物，以响应醛固酮和加压素。 开始，我们将定义AS 160的作用机制，AS 160是一种新鉴定的14-3-3结合蛋白，是醛固酮介导的ENaC运输的磷酸化依赖性调节剂。使用生物化学和功能测定，我们将测试的假设，AS 160稳定ENaC细胞内室在基础条件下，并允许ENaC运输到顶端膜响应其磷酸化。第二，我们将确定TBC 1D 1，一个新的目标确定的14-3-3亲和捕获，调节ENaC贩运响应加压素/PKA刺激。 这项工作有望揭示控制顶端ENaC密度的新机制，并确定治疗钠转运上皮细胞中盐和水平衡疾病的新靶点。 公共卫生相关性：这项名为“磷酸化依赖性调节上皮钠通道（ENaC）运输”的研究将研究磷酸化依赖性14-3-3结合蛋白是否稳定顶端ENaC运输途径中的多个步骤，以及它们的结合伴侣AS 160（TBC 1D 4）和TBC 1D 1是否是介导醛固酮和加压素作用的蛋白激酶的关键底物。通过这项研究，我们将进一步了解ENaC调节的分子机制，结果将进一步了解钠平衡，血容量和血压的控制。

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.