STRUCTURAL DETERMINANTS OF CFTR/SYNTAXIN INTERACTIONS

这个应用程序的主要目标是定义生理学 CFTRCl-调控的相关性和结构基础 通道受Synaxin 1A的影响。Synaxin 1A是一种膜蛋白，它是 在结肠上皮细胞中表达，定位于结肠上皮细胞 顶端细胞表面。Synaxin 1A与CFTR在物理上相互作用 当这些分子共同作用时，负调制CFTRc1-电流 在非洲爪哇卵母细胞中表达。我们未发表的结果表明 Synaxin 1A和CFTR之间的物理相互作用被抑制 PKA磷酸化和n-Sec1，一种突触素结合蛋白， 逆转合成素1A对cftr的负调控作用。我们 假设Synaxin 1A微调CFTRC1-电流活性 对生理线索的反应(例如，PKA激活)。具体目标 这项建议的内容如下。首先，我们将检验假设 CFTR-Synaxin 1A的相互作用受生理相关的调节 因子：即PKA、PKC和n-Sec1。之所以对PKC感兴趣，是因为它 使n-Sec1磷酸化并抑制其结合Synaxin 1A的能力；因此， 蛋白激酶C的激活可以从n-Sec1中释放Synaxin 1A，从而控制 针对CFTR的Synaxin 1A的可用性。其次，我们将定义 结合所需的CFTR和Syntxin 1A的最小结构域 并测试有缺陷的突变体的功能活性 有约束力的。我们的长期目标之一是在功能上活跃起来 不受Synaxin 1A负调控的cftr突变体 活着。第三，我们将检验CFTR和Synaxin 1A协同作用的假设 位于结肠上皮细胞顶端表面的分子复合体中 细胞。我们将确定Synaxin 1A是否与 表面标记的CFTR。我们还将确定CFTR是否驻留在 包括附加候选的多聚体复合体中的Synaxin 1A C1的调节器--电流活动。我们的研究结果应该是 为上皮细胞CFTR功能的调控提供新的见解 纸巾。

The broad goal of this application is to define the physiological relevance and the structural basis of the regulation of CFTR Cl- channels by syntaxin 1A. Syntaxin 1A is a membrane protein that is expressed in colonic epithelial cells where it localizes at or near the apical cell surface. Syntaxin 1A physically interacts with CFTR and negatively modulates CFTR C1- currents when these molecules are co- expressed in Xenopus oocytes. Our unpublished results indicate that the physical interaction between syntaxin 1A and CFTR is inhibited by PKA phosphorylation and by n-Sec1, a syntaxin-binding protein that reverses the negative modulation of CFTR by syntaxin 1A. We hypothesize that syntaxin 1A fine tunes CFTR C1- current activity in response to physiological clues (e.g., PKA activation). The specific aims of this proposal are as follows. First, we will test the hypothesis that the CFTR-syntaxin 1A interaction is regulated by physiologically relevant factors; namely PKA, PKC and n-Sec1. PKC is of interest because it phosphorylates n-Sec1 and inhibits its ability to bind syntaxin 1A; thus, PKC activation may release syntaxin 1A from n-Sec1 and thereby control the availability of syntaxin 1A for CFTR. Second, we will define the minimal domains of CFTR and syntxin 1A that are required for binding and test the functional activities of mutants that are defective at binding. One of our long-term goals is to generate functionally active CFTR mutants that cannot be negatively modulated by syntaxin 1A in vivo. Third, we will test the hypothesis that CFTR and syntaxin 1A co- reside in a molecular complex at the apical surfaces of colonic epithelial cells. We will determine if syntaxin 1A co-immunoprecipitates with surface-labeled CFTR. We will also determine if CFTR resides with syntaxin 1A in a multimeric complex that includes additional candidate regulators of C1- current activity. The results of our studies should provide new insights into the regulation of CFTR function in epithelial tissues.