ENaC Regulation by Cell Surface Associated SGK1

描述（由申请人提供）：收集管上皮钠通道（ENaC）活性对于维持细胞外液容量和血压至关重要。血清和糖皮质激素调节激酶1 （sgk1）已成为醛固酮信号通路的关键组成部分，刺激集管中的ENaC。研究最多的sgk1增加ENaC活性的机制涉及到顶端细胞膜通道数量的增加。然而，我们的实验室最近证明了sgk1也可以刺激ENaC的开放概率（Po），这代表了sgk1起作用的一种新方式。关于sgk1调控ENaC的机制，我们的知识仍然存在重大空白，包括以下问题：1)sgk1如何刺激ENaC Po，以及2)这种ENaC调控模式是否具有生理重要性。本研究的目的是确定sgk1刺激收集管中ENaC Po的新机制，并确定这种ENaC调节模式在体内是否重要。我们假设sgk1与ENaC相互作用并磷酸化ENaC以刺激通道Po。sgk1的这种作用机制不同于sgk1介导的Nedd4-2的调节，Nedd4-2是细胞表面ENaC表达的主要调节因子。我们提出三个具体目标。在Aim 1中，我们将测试sgk1是否独立于Nedd4-2刺激ENaC Po。我们将在sgk1或ENaC中引入以下突变：1)破坏nedd4 -2介导的ENaC调控；或2)破坏ENaC门控。然后我们将测试在这些条件下sgk1是否仍然可以增加通道Po。我们还将测试sgk1是否可以刺激Nedd4-2敲除小鼠的裂开收集管中的ENaC Po。在Aim 2中，我们将测试sgk1是否通过通道磷酸化刺激ENaC Po。我们将使用位点定向诱变来鉴定ENaC中被sgk1磷酸化的位点。然后我们将测试sgk1是否可以刺激这些突变通道的Po。在Aim 3中，我们将测试sgk1是否通过通道相互作用刺激ENaC Po。我们将使用位点定向诱变和阻断肽来确定sgk1中负责介导与ENaC相互作用的位点。我们将使用这些sgk1突变体和细胞渗透肽来破坏sgk1-ENaC相互作用，并测试这些试剂是否会损害sgk1介导的ENaC Po刺激和野生型小鼠收集管细胞和劈开收集管的活性。拟议的研究有望扩展sgk1作用的当前范式，并为sgk1如何刺激集管中的ENaC Po提供新的见解。我们期望这项研究将描述sgk1刺激ENaC Po的机制，并确定这种ENaC调节模式在体内的生理重要性。这项研究的影响可能为高血压的发病机制提供新的见解，并为治疗干预提供新的靶点（如sgk1-ENaC相互作用）。

DESCRIPTION (provided by applicant): Epithelial sodium channel (ENaC) activity in the collecting duct is essential for maintaining extracellular fluid volume and blood pressure. Serum and glucocorticoid regulated kinase 1 (sgk1) has emerged as a key component of the aldosterone signaling pathway that stimulates ENaC in the collecting duct. The most studied mechanism by which sgk1 increases ENaC activity involves an increase in the number of channels in the apical cell membrane. However, our laboratory has recently demonstrated that sgk1 can also stimulate the open probability (Po) of ENaC, which represents a novel means by which sgk1 acts. There remain critical gaps in our knowledge about the mechanisms that underlie sgk1 regulation of ENaC, including questions about: 1) how does sgk1 stimulate ENaC Po, and 2) whether this mode of ENaC regulation is physiologically important. The objective of the proposed research is to define novel mechanisms by which sgk1 stimulates ENaC Po in the collecting duct and determine whether this mode of ENaC regulation is important in vivo. We hypothesize that sgk1 interacts with and phosphorylates ENaC to stimulate channel Po. This mechanism of sgk1 action is distinct from sgk1-mediated regulation of Nedd4-2, a major regulator of cell surface ENaC expression. We propose three Specific Aims. In Aim 1, we will test whether sgk1 stimulates ENaC Po independent of Nedd4-2. We will introduce mutations in sgk1 or ENaC that: 1) disrupt Nedd4-2-mediated regulation of ENaC; or 2) disrupt ENaC gating. We will then test whether sgk1 can still increase channel Po under these conditions. We will also test whether sgk1 can stimulate ENaC Po in split-open collecting ducts from Nedd4-2 knockout mice. In Aim 2, we will test whether sgk1 stimulates ENaC Po through channel phosphorylation. We will use site- directed mutagenesis to identify sites in ENaC that are phosphorylated by sgk1. We will then test whether sgk1 can stimulate Po of these mutant channels. In Aim 3, we will test whether sgk1 stimulates ENaC Po through channel interaction. We will use site-directed mutagenesis and blocking peptides to identify sites in sgk1 responsible for mediating interaction with ENaC. We will use these sgk1 mutants and cell-permeant peptides to disrupt the sgk1-ENaC interaction and test whether these reagents will impair sgk1-mediated stimulation of ENaC Po and activity in collecting duct cells and split-open collecting ducts from wild type mice. The proposed research is expected to expand the current paradigm of sgk1 action and provide novel insights into how sgk1 stimulates ENaC Po in the collecting duct. We expect that this research will delineate the mechanisms by which sgk1 stimulates ENaC Po and establish the physiological importance of this mode of ENaC regulation in vivo. The impact of the proposed research may provide new insights into the pathogenesis of hypertension and suggest new targets (e.g. sgk1-ENaC interaction) for therapeutic intervention.