REGULATION OF CFTR BY APICAL MEMBRANE PKAII

顶端膜 PKAII 对 CFTR 的调节

• 批准号: 6661515
• 负责人: Monroe JACKSON Stutts
• 金额: $ 24.35万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6661515
• 关键词: apical membrane; cell component structure /function; chloride channels; cyclic AMP; cystic fibrosis; membrane permeability; membrane transport proteins; protein binding; protein kinase A; protein localization; respiratory epithelium; site directed mutagenesis; sodium channel

The major regulator of CFTR and ENaC in human airway epithelia is cAMP. In vivo, CFTR mediated Cl- conductance is approximately 50% active even at rest, and maximal activation can be attained when there is no detectable increase in total intracellular cAMP. These observations strongly suggest that protein kinase I (PKA) must be held within the apical plasma membrane in close proximity to CFTR and/or ENaC. Sequestration of PKA within very precise subcellular locations and in apposition with channels and transporter in not an unusual phenomenon. In fact, a diverse family of A- Kinase Anchoring Proteins (AKAPS) have been show to localize PKA in subcellular compartments in neurons, muscle and germ cells. Preliminary data for this application implicate AKAPS in the regulation of CFTR and ENaC in airway epithelia. Completion of these studies is Aim 1 of this application. However, the molecular identity of relevant AKAPS and their precise mechanisms of action promise enormous new insight into the regulation of apical membrane ion conductances. Therefore, in Aim 2, we will clone airway epithelial AKAPS by a well established and highly specific RII interaction strategy. In Aim 3 we will determine which airway epithelial AKAPS are concentrated at the apical cell membrane. We will use site-directed mutagenesis to identify RII binding sites and domains responsible for the apical localization. We will design cDNAs encoding mutant AKAPS that will exert dominant-negative effects when expressed in airway epithelial cell lines. These cell lines will be used to test the role of specific apical membrane AKAPS in the regulation of CFTR under basal and stimulated conditions. Finally, we will determine if the same apical membrane AKAPS that affect CFTR regulate ENaC activity or are involved in CFTR-mediated negative modulation of ENaC. The proposed studies will reveal how PKA is compartmentalized in the apical pole of airway epithelial cells, and will assess the significance of such localization for basal and stimulated function of apical membrane ion conductances.

人类气道上皮的CFTR和ENAC的主要调节器是营地。在 Vivo，CFTR介导的cl频率也约为50％，即使在 没有可检测到的休息和最大激活 总体内营地的增加。这些观察结果强烈暗示 该蛋白激酶I（PKA）必须保持在顶端质膜内 靠近CFTR和/或ENAC。 PKA在非常 精确的亚细胞位置以及带有通道和 在不寻常现象中的转运蛋白。实际上，一个多元化的家族 激酶锚定蛋白（AKAP）已显示以将PKA定位在 神经元，肌肉和生殖细胞中的亚细胞室。初步的 该应用程序的数据暗示了AKAP在CFTR和 ENAC在气道上皮中。这些研究的完成是目标1 应用。但是，相关Akap的分子身份及其 精确的行动机制有望对 调节顶膜离子电导。因此，在AIM 2中，我们 将通过一个良好且高度的克隆气道上皮Akap 特定的RII相互作用策略。在AIM 3中，我们将确定哪个气道 上皮AKAP集中在顶端细胞膜上。我们将使用 定向诱变以识别RII结合位点和域 负责顶端定位。我们将设计cdnas编码 突变的AKAP在表示时会产生显性阴性效应 气道上皮细胞系。这些细胞系将用于测试 特定顶端膜AKAP在CFTR调节中的作用 基础和刺激条件。最后，我们将确定是否相同 影响CFTR调节ENAC活性的顶膜AKAP或 参与CFTR介导的ENAC负面调节。提议 研究将揭示PKA如何在 气道上皮细胞，并将评估此类的重要性 顶膜离子基础和刺激功能的定位 电导。