Regulation of ENaC by Anionic Phospholipids

阴离子磷脂对 ENaC 的调节

• 批准号: 7257810
• 负责人: HE-PING MA
• 金额: $ 20.69万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7257810
• 关键词: 1-Phosphatidylinositol 3-Kinase; Affinity; Aldosterone; Amino Acids; Antibodies; Apical; Attenuated; Binding; Binding Sites; Biological Assay; Blood Pressure; Blood Volume; Cell surface; Cells; Charge; Confocal Microscopy; Cytoplasmic Tail; Data; Elevation; Epithelial; GRP gene; Homeostasis; Hormones; Immunoprecipitation; Injection of therapeutic agent; Kidney; Lasers; Lipids; Mediating; Membrane; Molecular; Mutate; Numbers; PH Domain; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phospholipids; Phosphoric Monoester Hydrolases; Physiological; Play; Point Mutation; Probability; Process; Proteins; Receptor Activation; Regulation; Role; Site; Sodium; Sodium Channel; Techniques; Testing; Transfection; arginyllysine; epithelial Na+ channel; intracellular protein transport; kinase inhibitor; patch clamp; phosphatidylinositol 3,4,5-triphosphate; protein transport; receptor; research study; steroid hormone

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 +稳态中起着非常重要的作用，从而控制全身血压。虽然ENaC的调节在过去十年中已被广泛研究，但膜磷脂对ENaC的调节仍在很大程度上未被探索。本申请中的初步数据表明，ENaC受阴离子磷脂如磷脂酰肌醇4，5-二磷酸（PIP 2）和磷脂酰肌醇3，4，5-三磷酸（PIP 3）的膜浓度调节。此外，在生理学上，ATP结合嘌呤能P2 Y受体似乎是阴离子磷脂的主要调节剂，从而是ENaC的调节剂。ATP通过磷脂酶C（PLC）的P2 v受体活化来抑制ENaC，磷脂酶C（PLC）降解PIPe，并且PIP 2的减少降低ENaC活性。除了P2 Y受体，ENaC的另一种生理调节剂，类固醇激素醛固酮，也改变阴离子磷脂的量。因此，我推测PIP 2和PIP 3可能介导ATP和醛固酮对ENaC的调节。该提议的第一个具体目标将集中于确定ATP是否通过降低PIP 2浓度来抑制ENaC以及醛固酮是否通过增加PIP 3浓度来刺激ENaC。ENaC活性的膜片钳研究将与具有特异性GFP融合的普列克底物蛋白同源结构域的膜PIP 2和PIP 3的激光共聚焦显微镜定位相关。由于β-和γ-ENaCs的胞质结构域含有赖氨酸和甘氨酸丰富的基序，我假设这些基序可能代表选择性的PIP 2和PIP 3结合位点。第二个具体目标将集中在确定是否截断和点突变的假定PIP 2和PIP 3结合位点废除ENaC的结合阴离子磷脂和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-蛋白质覆盖测定（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受体介导的ENaC调控的下游通路提供直接证据。