Regulation of ENaC by Anionic Phospholipids

阴离子磷脂对 ENaC 的调节

• 批准号: 7116378
• 负责人: HE-PING MA
• 金额: $ 21.31万
• 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/7116378
• 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 +稳态方面起着非常重要的作用，因此控制了全身血压。尽管在过去的十年中对ENAC的调节进行了广泛的研究，但膜磷脂对ENAC的调节仍然在很大程度上没有探索。本应用程序中的初步数据表明，ENAC受到阴离子磷脂的膜浓度的调节，例如磷脂酰氨基糖苷4,5-双磷酸（PIP2）和磷脂酰氨基糖苷3,4,5-三磷酸盐（PIP3）。此外，从生理上讲，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，从而是ENAC的调节剂。 ATP通过P2V受体激活磷脂酶C（PLC）抑制ENAC，该磷脂酶C（PLC）降低了管道，而PIP2的降低会降低ENAC活性。除了P2Y受体（ENAC的另一种生理调节剂）外，类固醇激素醛固酮还改变了阴离子磷脂的量。因此，我假设PIP2和PIP3可能会介导ATP和醛固酮对ENAC的调节。该提案的第一个具体目的将集中在确定ATP是否通过降低PIP2浓度抑制ENAC以及醛固酮是否通过增加PIP3浓度刺激ENAC。 ENAC活性的斑块夹研究将与膜PIP2和PIP3的激光共聚焦显微镜与特定的GFP融合的Pleckstrin同源域相关。由于β和γ-ENAC的细胞质结构域包含赖氨酸和精氨酸富基序，因此我假设这些基序可能代表选择性PIP2和PIP3结合位点。第二个特定目的将集中在确定假定的PIP2和PIP3结合位点的截断和点突变是否消除了ENAC与阴离子磷脂的结合以及ATP和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 - 蛋白质叠加测定（免疫沉淀）和激光共聚焦显微镜分析以及阴离子磷脂水平和ENAC细胞表面表达相关。这些研究将提供直接的证据，以进一步了解受体介导的ENAC调节的下游途径。