STRUCTURAL DETERMINANTS OF CFTR/SYNTAXIN INTERACTIONS

本申请的广泛目标是定义生理 相关性和CFTR Cl-调节的结构基础 syntaxin 1A的作用 Syntaxin 1A是一种膜蛋白， 在结肠上皮细胞中表达，其定位于或靠近 顶端细胞表面 突触融合蛋白1A与CFTR物理相互作用， 负调节CFTR C1-电流时，这些分子是共同的， 在非洲爪蟾卵母细胞中表达。 我们未发表的结果表明， 突触融合蛋白1A和CFTR之间的物理相互作用被抑制， PKA磷酸化和n-Sec 1，一种突触融合蛋白结合蛋白， 逆转突触融合蛋白1A对CFTR的负调节。 我们 假设突触融合蛋白1A微调CFTR C1电流活性， 对生理线索的响应（例如，PKA活化）。 具体目标 这一建议的内容如下。 首先，我们将测试假设， CFTR-突触融合蛋白1A相互作用受生理相关的 蛋白激酶A、蛋白激酶C和n-Sec 1。 PKC很有意义，因为它 磷酸化n-Sec 1并抑制其结合突触融合蛋白1A的能力;因此， PKC激活可从n-Sec 1释放syntaxin 1A，从而控制 突触融合蛋白1A对CFTR的可用性。 其次，我们将定义 CFTR和syntxin 1A结合所需的最小结构域 并测试突变体的功能活性， 约束力 我们的长期目标之一是产生功能活跃的 CFTR突变体不能被syntaxin 1A负调节， vivo. 第三，我们将测试CFTR和syntaxin 1A共同作用的假设。 存在于结肠上皮顶端表面的分子复合物中 细胞 我们将确定突触融合蛋白1A是否与 表面标记的CFTR。 我们还将确定CFTR是否与 多聚体复合物中的突触融合蛋白1A C1电流活动的调节器。 我们的研究结果应该 为上皮细胞CFTR功能的调节提供了新的见解， 组织中

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 Our unpublished results indicate that the physical interaction between syntaxin 1A and CFTR is inhibited by PKA phosphorylation and by n-Sec1, a syntaxin-binding protein that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.