Regulation of ENaC by Anionic Phospholipids

阴离子磷脂对 ENaC 的调节

• 批准号: 6894027
• 负责人: HE-PING MA
• 金额: $ 21.79万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6894027
• 关键词: Xenopus; Xenopus oocyte; adenosine triphosphate; aldosterone; anions; binding sites; calcium ion; confocal scanning microscopy; electrolyte balance; immunoprecipitation; lipid metabolism; membrane activity; phosphatidylinositols; phospholipase C; podocyte; point mutation; protein kinase C; protein structure; sodium channel; voltage /patch clamp

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.

描述（由申请人提供）：肾上皮钠通道（ENaC）在维持Na + 稳态方面发挥着非常重要的作用，从而控制全身血压。尽管在过去十年中，ENaC 的调节已被广泛研究，但膜磷脂对 ENaC 的调节仍然很大程度上未被探索。本申请的初步数据表明，ENaC 受阴离子磷脂（例如磷脂酰肌醇 4,5-二磷酸 (PIP2) 和磷脂酰肌醇 3,4,5-三磷酸 (PIP3)）的膜浓度调节。此外，从生理学角度来看，与嘌呤能 P2Y 受体结合的 ATP 似乎是阴离子磷脂的主要调节剂，从而也是 ENaC 的调节剂。 ATP 通过 P2v 受体激活磷脂酶 C (PLC) 来降解 PIPe，从而抑制 ENaC，并且 PIP2 的减少会降低 ENaC 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量。因此，我推测PIP2和PIP3可能介导ATP和醛固酮对ENaC的调节。该提案的第一个具体目标将集中于确定 ATP 是否通过降低 PIP2 浓度来抑制 ENaC，以及醛固酮是否通过增加 PIP3 浓度来刺激 ENaC。 ENaC 活性的膜片钳研究将与具有特定 GFP 融合的 pleckstrin 同源域的膜 PIP2 和 PIP3 的激光共聚焦显微镜定位相关。由于 β-和 γ-ENaC 的细胞质结构域含有富含赖氨酸和精氨酸的基序，我假设这些基序可能代表选择性 PIP2 和 PIP3 结合位点。第二个具体目标将集中于确定假定的 PIP2 和 PIP3 结合位点的截短和点突变是否会消除 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。电生理学研究将与脂质-蛋白质叠加测定（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 ENaC 受体介导的调节下游途径提供直接证据。