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: 8389828
• 负责人: Abdel Ayube Alli
• 金额: $ 5.22万
• 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/8389828
• 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.

描述（由申请人提供）：上皮钠通道（ENAC）在维持钠平衡，血容量和血压方面起着重要作用。它位于各种上皮细胞的根尖膜，包括那些沿肾远端肾单位的细胞。先前的观察结果表明，肌动蛋白细胞骨架在ENAC活性的调节中起作用，并且众所周知，ENAC受磷酸肌醇的调节（即PIP2和PIP3）。然而，肌动蛋白细胞骨架调节ENAC的机制以及如何将这些稀有磷酸肌醇提交给ENAC的机制尚不清楚。该提案描述了研究肌动蛋白细胞骨架是MARCKS，钙调蛋白和ENAC的组织中心的实验，并且该组织对于通过磷酸肌醇的MARCKS依赖性调节ENAC活性是必不可少的。我们将使用各种分子和蛋白质组学技术（例如，共免疫沉淀研究，GST下拉测定法和荧光谐振能传递（FRET））来识别与磷酸固醇相关的磷酸固醇相关蛋白的细胞骨架相关蛋白，以确定eNAC的依赖性和依赖于ENAC的依赖于eNAC的差异化磷酸化的磷酸化磷酸化。我们将使用特定药理剂的电生理方法来确定MARCKS和磷酸肌醇在调节异爪蟾远端肾小管上皮细胞中表达内源性eNAC的顶膜ENAC活性中的作用。我们将在光漂白后进行荧光恢复（FRAP），以确定肌动蛋白细胞骨架在MARCKS，钙调蛋白和ENAC的膜信号复合物形成中的作用。这项研究的总体目的是了解ENAC受肌动蛋白细胞骨架，MARCKS和磷酸肌醇调节的机制，并更好地了解健康和疾病中的ENAC的控制。