Regulation of ENaC by phosphoinositides, MARCKS, and the cytoskeleton

描述(申请人提供)：上皮性钠通道(ENaC)在维持钠平衡、血容量和血压方面起着重要作用。它定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活性的调节中起作用，已知ENaC受磷脂酰肌醇(PIP2和PIP3)的调节。然而，肌动蛋白细胞骨架调节ENaC的机制以及这些稀有的磷脂酰肌醇是如何被提呈给ENaC的还不清楚。这项建议描述了实验，以调查这一假设，即肌动蛋白细胞骨架作为Marcks，钙调蛋白和ENaC的组织中心，以及这种组织对于Marcks依赖的磷酸肌醇调节ENaC活性是必要的。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下拉试验和荧光共振能量转移(FRET))来识别参与ENaC的肌醇磷脂依赖调控的细胞骨架相关蛋白质，并确定哪些ENaC结构域与肌醇磷脂有差异地结合。我们将使用电生理方法结合特定的药理药物来确定Marcks和磷酸肌醇在表达内源性ENaC的非洲爪哇远端肾单位上皮细胞中对顶膜ENaC活性的调节作用。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膜信号复合体形成中的作用。这项研究的总体目标是了解ENaC受肌动蛋白细胞骨架、Marcks和磷脂酰肌醇调节的机制，并更好地了解ENaC在健康和疾病中的控制。

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.