Regulation of ENaC by phosphoinositides, MARCKS, and the cytoskeleton

磷酸肌醇、MARCKS 和细胞骨架对 ENaC 的调节

• 批准号: 8203348
• 负责人: Abdel Ayube Alli
• 金额: $ 4.84万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8203348
• 关键词: Actins; Binding; Biological Assay; Blood Pressure; Blood Volume; C-terminal; Calmodulin; Cells; Chimeric Proteins; Co-Immunoprecipitations; Complex; Cytochalasins; Cytoplasm; Cytoskeleton; Disease; Distal; Drug Delivery Systems; Epithelial; Epithelial Cells; Equilibrium; F-Actin; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Goals; Health; Heart Diseases; Homeostasis; Hypertension; In Vitro; Investigation; Kidney; Kidney Failure; Lateral; Lead; MARCKS gene; Mass Spectrum Analysis; Measures; Membrane; Methods; Molecular; N-terminal; Nephrons; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Play; Production; Proteins; Proteomics; Recombinants; Regulation; Research Proposals; Role; Signal Transduction; Sodium; Sodium Channel; Sodium Chloride; Spectrin; Stroke; Techniques; Two-Dimensional Gel Electrophoresis; United States; Water; Xenopus; apical membrane; blood pressure regulation; crosslink; epithelial Na+ channel; extracellular; gamma ENaC; hypertension control; novel; polymerization; prevent; research study

DESCRIPTION (provided by applicant): The epithelial sodium channel (ENaC) plays an important role in maintaining sodium balance, blood volume, and blood pressure. It is localized to the apical membrane of various epithelial cells including those that line the distal renal nephron. Prior observations suggest that the actin cytoskeleton plays a role in the regulation of ENaC activity and it is known that ENaC is regulated by phosphoinositides (i.e. PIP2 and PIP3). However, the mechanism by which the actin cytoskeleton regulates ENaC and how these rare phosphoinositides are presented to ENaC is unknown. This proposal describes experiments to investigate the hypothesis that the actin cytoskeleton serves as an organizing center for MARCKS, calmodulin, and ENaC and that this organization is necessary for MARCKS-dependent regulation of ENaC activity by phosphoinositides. We will use various molecular and proteomic techniques (e.g., co-immunoprecipitation studies, GST pull-down assays, and fluorescence resonance energy transfer (FRET)) to identify cytoskeletal-associated proteins involved in the phosphoinositide-dependent regulation of ENaC and to determine which ENaC domains differentially bind phosphoinositides. We will use electrophysiological methods with specific pharmacological agents to determine the role of MARCKS and phosphoinositides in regulating apical membrane ENaC activity in Xenopus distal nephron epithelial cells expressing endogenous ENaC. We will perform Fluorescence Recovery after Photobleaching (FRAP) to determine the role of the actin cytoskeleton in the formation of a membrane signaling complex of MARCKS, calmodulin, and ENaC. The overall goal of this investigation is to understand the mechanism by which ENaC is regulated by the actin cytoskeleton, MARCKS, and phosphoinositides and to gain a better understanding for the control of ENaC in health and disease. PUBLIC HEALTH RELEVANCE: Hypertension or high blood pressure is the leading cause of stroke, heart disease, and kidney failure in the United States. The epithelial sodium channel (ENaC) plays a critical role in blood pressure regulation by controlling salt and water homeostasis. This research proposal is intended to investigate the mechanism by which ENaC is regulated by the actin cytoskeleton, MARCKS, and phosphoinositides. If the specific aims of this project are accomplished it would allow for a better understanding of how ENaC is regulated, and it may lead to novel drug targets for preventing and/or controlling hypertension.

描述（申请人提供）：上皮钠通道（ENaC）在维持钠平衡、血容量和血压方面发挥着重要作用。它位于各种上皮细胞的顶膜，包括那些排列在远端肾单位的上皮细胞。先前的观察表明，肌动蛋白细胞骨架在 ENaC 活性的调节中发挥作用，并且已知 ENaC 受磷酸肌醇（即 PIP2 和 PIP3）调节。然而，肌动蛋白细胞骨架调节 ENaC 的机制以及这些罕见的磷酸肌醇如何呈递给 ENaC 尚不清楚。该提案描述了研究以下假设的实验：肌动蛋白细胞骨架作为 MARCKS、钙调蛋白和 ENaC 的组织中心，并且该组织对于磷酸肌醇对 ENaC 活性的 MARCKS 依赖性调节是必需的。我们将使用各种分子和蛋白质组学技术（例如，免疫共沉淀研究、GST 下拉分析和荧光共振能量转移 (FRET)）来鉴定参与 ENaC 磷酸肌醇依赖性调节的细胞骨架相关蛋白，并确定哪些ENaC 结构域与磷酸肌醇的结合存在差异。我们将使用特定药物的电生理学方法来确定 MARCKS 和磷酸肌醇在表达内源性 ENaC 的爪蟾远端肾单位上皮细胞中调节顶膜 ENaC 活性中的作用。我们将进行光漂白后荧光恢复 (FRAP)，以确定肌动蛋白细胞骨架在 MARCKS、钙调蛋白和 ENaC 膜信号复合物形成中的作用。本研究的总体目标是了解肌动蛋白细胞骨架、MARCKS 和磷酸肌醇调节 ENaC 的机制，并更好地了解 ENaC 在健康和疾病中的控制。 公共卫生相关性：在美国，高血压是中风、心脏病和肾衰竭的主要原因。上皮钠通道 (ENaC) 通过控制盐和水的稳态在血压调节中发挥着关键作用。本研究计划旨在研究肌动蛋白细胞骨架、MARCKS 和磷酸肌醇调节 ENaC 的机制。如果该项目的具体目标得以实现，将有助于更好地了解 ENaC 的调节方式，并且可能会产生预防和/或控制高血压的新药物靶点。