REGULATION OF CFTR BY APICAL MEMBRANE PKAII

顶端膜 PKAII 对 CFTR 的调节

• 批准号: 6496771
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6496771
• 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.

人气道上皮细胞中 CFTR 和 ENaC 的主要调节因子是 cAMP。在 体内，CFTR 介导的 Cl- 电导率约为 50%，即使在 休息，当没有可检测到的情况下可以达到最大激活 细胞内总 cAMP 增加。这些观察强烈表明 蛋白激酶 I (PKA) 必须位于顶端质膜内 靠近 CFTR 和/或 ENaC。 PKA 在非常内的隔离 精确的亚细胞位置并与通道并置 运输者并不是什么不寻常的现象。事实上，A 的多元化家庭 激酶锚定蛋白 (AKAPS) 已被证明可以将 PKA 定位于 神经元、肌肉和生殖细胞中的亚细胞区室。初步的 该应用程序的数据表明 AKAPS 参与了 CFTR 的监管，并且 气道上皮细胞中的 ENaC。完成这些研究是本次会议的目标 1 应用。然而，相关 AKAPS 的分子特性及其 精确的作用机制有望为我们带来巨大的新见解 顶膜离子电导的调节。因此，在目标 2 中，我们 将通过成熟且高度成熟的方法克隆气道上皮 AKAPS 具体的RII交互策略。在目标 3 中，我们将确定哪个气道 上皮 AKAPS 集中在顶端细胞膜上。我们将使用 定点诱变以鉴定 RII 结合位点和结构域 负责心尖定位。我们将设计cDNA编码 突变型 AKAPS 在表达时会产生显性负效应 气道上皮细胞系。这些细胞系将用于测试 特定顶膜 AKAPS 在 CFTR 调节中的作用 基础条件和刺激条件。最后我们判断是否相同 影响 CFTR 的顶膜 AKAPS 调节 ENaC 活性或 参与 CFTR 介导的 ENaC 负调节。拟议的 研究将揭示 PKA 如何在心尖极区分开 气道上皮细胞，并将评估这种意义 顶膜离子的基础和刺激功能的定位 电导。