Regulation of ENaC by Anionic Phospholipids

描述(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，从而控制全身血压。尽管在过去的十年中，对ENaC的调节已经得到了广泛的研究，但膜磷脂对ENaC的调节在很大程度上仍未被探索。应用中的初步数据表明，ENaC受阴离子磷脂如磷脂酰肌醇4，5-二磷酸(PIP2)和磷脂酰肌醇3，4，5-三磷酸(PIP3)的膜浓度调节。此外，生理上，与嘌呤能P2Y受体结合的ATP似乎是阴离子磷脂的主要调节因子，从而是ENaC的调节因子。ATP通过P2V受体激活降解PIPE的磷脂酶C(PLC)来抑制ENaC，PIP2的减少降低了ENaC的活性。除了P2Y受体，ENaC的另一个生理调节剂，类固醇激素醛固酮，也改变了阴离子磷脂的数量。因此，我推测PIP2和PIP3可能介导了ATP和醛固酮对ENaC的调节。该提案的第一个具体目标将集中在确定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。膜片钳对ENaC活性的研究将与激光共聚焦显微镜对具有特定GFP融合Pleckstrin同源结构域的膜PIP2和PIP3的定位相关。由于β-和γ-ENaC的细胞质结构域包含富含赖氨酸和精氨酸的基序，我推测这些基序可能代表选择性的PIP2和PIP3结合位点。第二个具体目标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取消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-蛋白质覆盖分析(免疫沉淀)和激光共聚焦显微镜分析阴离子磷脂水平和ENaC细胞表面表达相关联。这些研究将为进一步了解受体介导的ENaC调节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.