Traffic Regulatory Proteins and ENaC

交通调节蛋白和 ENaC

• 批准号: 8305037
• 负责人: RAYMOND A FRIZZELL
• 金额: $ 31.12万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305037
• 关键词: 14-3-3 Proteins; Accounting; Actins; Address; Affinity; Agonist; Aldosterone; Apical; Attention; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Blood Pressure; Cell surface; Cells; Chimeric Proteins; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis; Data; Distal; Duct (organ) structure; Ductal Epithelium; Endocytosis; Environment; Epithelial; Epithelial Cells; Epithelium; Equilibrium; Event; Exocytosis; Extracellular Fluid; Forskolin; Funding; GTPase-Activating Proteins; Hormonal; Hypertension; Insulin; Kidney; Kidney Diseases; Knockout Mice; Knowledge; Life; Link; Liquid substance; Location; Lung diseases; Mediating; Mediator of activation protein; Membrane; Membrane Protein Traffic; Methods; Nephrons; Nephrosis; Pathway interactions; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physical condensation; Physiological; Probability; Process; Protein Isoforms; Protein Kinase; Proteins; Proteomics; Publishing; Pulmonary Edema; Pulmonary Hypertension; Recycling; Regulation; Research; Retrieval; Role; Signal Transduction; Sodium; Sodium Channel; Sodium Chloride; Stimulus; Surface; Total Internal Reflection Fluorescent; Universities; Vasopressins; Water; Work; airway epithelium; apical membrane; base; cellular imaging; cofilin; density; deprivation; epithelial Na+ channel; genetic regulatory protein; hormone regulation; human disease; in vivo Model; mutant; novel; novel strategies; public health relevance; rab GTP-Binding Proteins; renal epithelium; response; synergism; therapeutic target; tool; trafficking

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.

描述（由申请人提供）：钠穿过肾和气道上皮的管腔膜进入是一个高度调控的过程，由上皮钠通道（ENaC）介导。 ENaC 功能异常与重大人类疾病有关，包括高血压、肾病、囊性纤维化和肺水肿。在肾和气道上皮细胞中，ENaC 活性受到控制顶膜中活性通道数量的因素的调节。顶端通道数量由膜运输事件决定，膜运输事件响应细胞外液量和血压的关键激素调节剂。该提案解决了肾上皮细胞中 ENaC 运输的磷酸化依赖性调节。该领域的几乎所有研究都集中在控制从顶膜回收 ENaC 的机制上。相比之下，我们对调节 ENaC 向前运输至顶端细胞表面的机制的了解还很薄弱。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正向运输中重要调节节点处的蛋白质的工具。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。首先，我们将定义 Rab-GAP、AS160、14-3-3 结合蛋白以及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。一种相关蛋白 TBC1D1 是响应加压素刺激的顶端 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作用的协同作用。该方法还鉴定了肌动蛋白重组蛋白 14-3-3 结合蛋白 cofilin，作为控制受调节的顶端 ENaC 插入的候选蛋白。我们的工作预计将揭示控制顶端 ENaC 密度的新机制，并确定钠转运上皮细胞中盐和水平衡异常的治疗靶点。 公共健康相关性：该提案旨在确定肾上皮细胞顶膜上皮钠通道 (ENaC) 密度的关键调节因子。 ENaC 运输到顶端表面是感知细胞外液容量和血压的激素通道调节的主要模式。这些途径与重要的人类疾病有关，包括高血压。使用 14-3-3 亲和方法，我们已经确定了几种新的 ENaC 正向运输至顶膜的调节因子，并且我们将定义它们响应激动剂控制沿顶端 ENaC 运输途径的重要步骤的机制。这项工作有望揭示控制肾脏中钠和液体转运的新调节因子和治疗靶点。