Regulation of ENaC by Anionic Phospholipids

描述（由申请人提供）：肾上皮钠通道（ENaC）在维持Na +稳态中起着非常重要的作用，从而控制全身血压。尽管在过去的十年中ENaC的调控已被广泛研究，但膜磷脂对ENaC的调控在很大程度上仍未被探索。本应用的初步数据表明，ENaC受阴离子磷脂的膜浓度调节，如磷脂酰亚油醇4,5-二磷酸（PIP2）和磷脂酰亚油醇3,4,5-三磷酸（PIP3）。此外，在生理上，ATP结合嘌呤能P2Y受体似乎是阴离子磷脂的主要调节剂，因此也是ENaC的调节剂。ATP通过P2v受体激活降解PIPe的磷脂酶C （PLC）抑制ENaC， PIP2的减少降低ENaC的活性。除了P2Y受体外，ENaC的另一种生理调节剂，类固醇激素醛固酮，也会改变阴离子磷脂的数量。因此，我推测PIP2和PIP3可能介导ATP和醛固酮对ENaC的调节。本提案的第一个具体目标将集中于确定ATP是否通过降低PIP2浓度抑制ENaC，醛固酮是否通过增加PIP3浓度刺激ENaC。膜片钳研究ENaC活性将与激光共聚焦显微镜定位具有特定gfp融合的pleckstrin同源结构域的PIP2和PIP3膜相关。由于β -和γ - enacs的细胞质结构域含有赖氨酸和精氨酸丰富的基序，我假设这些基序可能代表了PIP2和PIP3的选择性结合位点。第二个具体目标将集中于确定假定的PIP2和PIP3结合位点的截断和点突变是否会破坏ENaC与阴离子磷脂的结合，以及ATP和醛固酮对ENaC活性和细胞表面表达的影响。电生理研究将与脂质-蛋白覆盖试验（免疫沉淀）和阴离子磷脂水平和ENaC细胞表面表达的激光共聚焦显微镜分析相关。这些研究将为进一步了解受体介导的ENaC调控的下游途径提供直接证据。

DESCRIPTION (provided by applicant): The renal epithelial sodium channel (ENaC) plays a very important role in maintaining Na + homeostasis, and consequently controls systemic blood pressure. Although the regulation of ENaC has been extensively studied during the past decade, the regulation of ENaC by membrane phospholipids remains largely unexplored. Preliminary data in this application have shown that ENaC is regulated by the membrane concentration of anionic phospholipids such as phosphatidylinosotol 4,5-bisphosphate (PIP2) and phosphatidylinosotol 3,4,5- trisphosphate (PIP3). In addition, physiologically, ATP binding to purinergic P2Y receptors appears to be a primary regulator of anionic phospholipids and, thereby, a regulator of ENaC. ATP inhibits ENaC by P2v receptor activation of phospholipase C (PLC) that degrades PIPe, and the reduction in PIP2 reduces ENaC activity. Besides P2Y receptors, another physiological regulator of ENaC, the steroid hormone aldosterone, also alters the amount of anionic phospholipids. Therefore, I hypothesize that PIP2 and PIP3 might mediate the regulation of ENaC by ATP and aldosterone. The first specific aim of this proposal will be focused on determining whether ATP inhibits ENaC by decreasing PIP2 concentration and whether aldosterone stimulates ENaC by increasing PIP3 concentration. Patch-clamp studies of ENaC activity will be correlated to laser confocal microscopy localization of membrane PIP2 and PIP3 with specific GFP-fused pleckstrin homology domains. Since the cytoplasmic domains of beta- and gamma-ENaCs contain lysine- and arginine-rich motifs, I hypothesize that these motifs might represent selective PIP2 and PIP3 binding sites. The second specific aim will be focused on determining whether truncations and point mutations of the putative PIP2 and PIP3 binding sites abolish both the binding of ENaC to anionic phospholipids and the effects of ATP and aldosterone on ENaC activity and cell surface expression. Electrophysiological studies will be correlated to both lipid-protein overlay assays (immunoprecipitation) and laser confocal microscopy analysis of anionic phospholipids levels and ENaC cell surface expression. These studies will provide direct evidence for further understanding the downstream pathways for receptor-mediated regulation of ENaC.