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.

气道上皮的Na+吸收通过调节气道表面液体（ASL）的深度来影响粘膜清除率（MCC）。过多的Na+吸收降低，抑制Na+吸收会刺激MCC。顶部膜中的Amiloride敏感上皮Na+通道（ENAC）是Na+吸收的速率限制步骤。尽管ENAC在许多组织中都表达并进行了充分的研究，但大多数研究都集中在盐水中盐的荷尔蒙刺激上。气道中的Na+吸收在盐保护中没有作用，并且不受刺激ENAC的激素影响。取而代之的是，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。这种效果预测了将局部刺激与ENAC活性联系起来的信号通路，但是有关这些转导机制的知识是有限的。 ENAC的三个同源亚基中的每个 在其胞质氨基末端包含一个高度保守的门控域。我们假设ASL中存在的抑制性刺激可通过与ENAC氨基末端相互作用的途径降低Na+吸收，从而降低ENAC开放概率。我们将对ASL中核苷酸的情况检验该假设。已知核苷酸可以激活磷脂酶C（PLCBETA）的β同工酶。在AIM 1中，我们将测试PLCBETA活性在ENAC调控气道上皮细胞中ENAC中的作用，我们将确定顶端膜中特定的PLCBETA同工酶（S）并确定PLC蛋白结合伴侣。在AIM 2中，我们将检验以下假设：PLC底物4,5磷酸磷酸磷酸（PIP2）与ENAC氨基末端结合并通过识别特定残基来稳定ENAC氨基末端，并稳定ENAC开放构象。 与PIP2和其他阴离子磷脂相互作用。最后，在AIM 3中，我们将使用一种蛋白质组学方法来识别与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PIP2与ENAC氨基末端结合的影响。