Regulation of ENaC by phosphoinositides, MARCKS, and the cytoskeleton

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重要作用。它局限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，并且已知ENaC受磷酸肌苷（即PIP2和PIP3）的调节。然而，肌动蛋白细胞骨架调节ENaC的机制以及这些罕见的磷酸肌苷如何呈现给ENaC尚不清楚。该提案描述了研究肌动蛋白细胞骨架作为MARCKS、钙调蛋白和ENaC的组织中心的假设的实验，并且该组织对于磷酸肌苷对ENaC活性的marks依赖性调节是必要的。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下拉试验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差异结合。我们将使用电生理方法和特定药物来确定MARCKS和磷酸肌苷在调节表达内源性ENaC的爪蟾远端肾元上皮细胞顶膜ENaC活性中的作用。我们将进行光漂白后荧光恢复（FRAP）来确定肌动蛋白细胞骨架在marks、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ENaC受肌动蛋白细胞骨架、marks和磷酸肌苷调节的机制，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.