MOLECULAR MECHANISMS OF LOCAL ENAC REGULATION

当地 ENAC 监管的分子机制

• 批准号: 6809808
• 负责人: Monroe JACKSON Stutts
• 金额: $ 21.5万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-16 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6809808
• 关键词: 3T3 cells; SDS polyacrylamide gel electrophoresis; Xenopus oocyte; affinity chromatography; apical membrane; body water dehydration; chloride channels; cystic fibrosis; electrolyte balance; electrophysiology; enzyme activity; enzyme induction /repression; immunocytochemistry; immunoprecipitation; nucleotides; phosphatidylinositols; phospholipase C; phospholipids; posttranslational modifications; protein binding; protein protein interaction; protein structure function; proteomics; respiratory epithelium; sodium; tissue /cell culture

Na+ absorption by airway epithelia influences the rate of mucociliary clearance (MCC) by adjusting the depth of airway surface liquid (ASL). Excessive Na+ absorption decreases, and inhibited Na+ absorption stimulates MCC. The amiloride sensitive epithelial Na+ channel (ENaC) in the apical membrane is the rate-limiting step in Na+ absorption. Although ENaC is expressed in many tissues and is well studied, most investigations have focused on hormonal stimulation of ENaC in salt conserving epithelia. Na+ absorption in airways plays no role in salt conservation and is unaffected by hormones that stimulate ENaC. Instead, signals present in ASL, such as nucleotides, typically increase the depth of ASL and the rate of MCC by inhibiting Na+ absorption. This effect predicts signaling pathways that link local stimuli to ENaC activity, but knowledge about these transduction mechanisms is limited. Each of ENaC's three homologous subunits contains a highly conserved gating domain in its cytosolic amino terminus. We hypothesize that inhibitory stimuli present in ASL decrease Na+ absorption through pathways that interact with ENaC amino termini to decrease ENaC open probability. We will test this hypothesis for the case of nucleotides in ASL. Nucleotides are known to activate the beta isozymes of phospholipase C (PLCbeta). In Aim 1, we will test the role of PLCbeta activity in the regulation of ENaC in the apical membrane of airway epithelial cells and we will identify the specific PLCbeta isozyme(s) in the apical membrane and determine PLC protein-binding partners. In Aim 2 we will test the hypothesis that the PLC substrate, 4,5 phosphoinositide phosphate (PIP2), binds to the ENaC amino termini and stabilizes the ENaC open conformation by identifying the specific residues that interact with PIP2 and other anionic phospholipids. Finally, in Aim 3 we will use a proteomic approach to identify proteins that interact with the amino termini of ENaC. We will determine if these interactions regulate ENaC gating and are affected by PIP2 binding to the ENaC amino termini.

气道上皮对 Na+ 的吸收通过调节气道表面液体 (ASL) 的深度来影响粘膜纤毛清除率 (MCC)。过多的Na+吸收减少，抑制Na+吸收会刺激MCC。顶膜中的阿米洛利敏感上皮 Na+ 通道 (ENaC) 是 Na+ 吸收的限速步骤。尽管 ENaC 在许多组织中表达并且得到了充分研究，但大多数研究都集中在盐保存上皮细胞中 ENaC 的激素刺激上。气道中的 Na+ 吸收对盐保存没有作用，并且不受刺激 ENaC 的激素的影响。相反，ASL 中存在的信号（例如核苷酸）通常通过抑制 Na+ 吸收来增加 ASL 的深度和 MCC 的速率。这种效应预测了将局部刺激与 ENaC 活性联系起来的信号通路，但有关这些转导机制的知识有限。 ENaC 的三个同源亚基中的每一个 在其胞质氨基末端含有高度保守的门控结构域。我们假设 ASL 中存在的抑制性刺激通过与 ENaC 氨基末端相互作用的途径减少 Na+ 吸收，从而降低 ENaC 开放概率。我们将用 ASL 中核苷酸的情况来检验这一假设。已知核苷酸可激活磷脂酶 C (PLCbeta) 的 β 同工酶。在目标 1 中，我们将测试 PLCbeta 活性在气道上皮细胞顶膜 ENaC 调节中的作用，我们将鉴定顶膜中特定的 PLCbeta 同工酶并确定 PLC 蛋白结合伴侣。在目标 2 中，我们将测试以下假设：PLC 底物 4,5 磷酸肌醇磷酸 (PIP2) 与 ENaC 氨基末端结合，并通过识别特定残基来稳定 ENaC 开放构象。 与 PIP2 和其他阴离子磷脂相互作用。最后，在目标 3 中，我们将使用蛋白质组学方法来鉴定与 ENaC 氨基末端相互作用的蛋白质。我们将确定这些相互作用是否调节 ENaC 门控并受到 PIP2 与 ENaC 氨基末端结合的影响。