CFTR-DEPENDENT MEMBRANE RECYCLING AND CI TRANSPORT

该项目的长期目标是确定分子 调节交通和功能活动的机制 CFTR CL通道位于上皮细胞表面。 此续订 应用集中在两个CFTR功能的调节 膜运输调节因子的突触融合蛋白家族成员 (i.e.,突触融合蛋白1A和3）;其中每一个都在顶端表达 结肠上皮细胞的两极。 突触融合蛋白1A 与CFTR CI通道，并调节CFTR CL电流， 结肠上皮细胞和异源表达系统。 我们 提出snytaxin 1A调节CFTR功能， （二）通过调整CFTR的数量 细胞表面的分子，作为控制 CFTR的细胞内运输和/或（ii）通过直接调节 CFTR Cl通道通过蛋白质-蛋白质相互作用。 这项建议 将通过追求三个具体目标进行测试。 首先，我们将核实 突触融合蛋白1A调节CFTR CI电流活性， 确定sntaxin IA对CFTR CI电流的调节是否 与CFTR分子数量的变化相关， 细胞表面 我们还将确定syntaxin 1A是否直接 调节切除的膜斑中的CFTR Cl通道， 平面脂质双层。 第二个具体目标是确定 突触融合蛋白1A之间物理相互作用的结构基础 和CFTR。 我们将绘制每个区域的相关结合位点， 这些分子和表征的功能活动， 缺乏结合CFTR能力的突触融合蛋白1A突变体。 的 通过n-Sec 1调节这种相互作用（即，突触融合蛋白1A 也在结肠上皮细胞中表达的结合蛋白） 和cAMP依赖性蛋白激酶（磷酸化 两种分子）也将被检查。 第三个目标是 定义syntaxins 1A和3在 控制顶端膜运输（例如，内吞作用和 膜再循环）。 我们 这些结果将为分子机制提供新的见解 控制CFTR CI的流量和功能活动 渠道，这是有缺陷的或缺乏在最常见的 高加索人的遗传性疾病（即囊性纤维化）。

The long-term objective of this project is to define the molecular mechanisms that regulate the traffic and functional activity of CFTR CL channels at the epithelial cell surface. This renewal application focus on the regulation of CFTR function by two members of the syntaxin family of membrane traffic regulators (i.e., syntaxins 1A and 3); each of which is expressed at the apical poles of colonic epithelial cells. Syntaxin 1A physically associates with CFTR CI channels and regulates CFTR CL currents in colonic ephithelial cells and heterologous expression systems. We propose that snytaxin 1A regulates CFTR function in one or both of the following ways: (ii) by regulating the numbers of CFTR molecules at the cell surface as part of the machinery that controls the intracellular traffic of CFTR and/or (ii) by directly modulating CFTR CI channels via protein-protein interactions. This proposal will be tested by pursuing 3 specific aims. First, we will verify that syntaxin 1A modulates CFTR CI current activity and determine if the regulation of CFTR CI currents by sntaxin 1 A correlates with changes in the numbers of CFTR molecules at the cell surface. We will also determine if syntaxin 1A directly regulates CFTR CI channels in excised membrane patches and planar lipid bilayers. The second specific aim is to define the structural basis for the physical interaction between syntaxin 1A and CFTR. We will map the relevant binding sites on each of these molecules and characterize the functional activities of syntaxin 1A mutants that lack the ability to bind CFTR. The regulation of this interaction by n-Sec 1 (i.e., a syntaxin 1A binding protein that I s also expressed in colonic epithelial cells) and by cAMP-dependent protein kinase (which phosphorylates both molecules) will be also be examined. the third aim is to define the specific roles that syntaxins 1A and 3 play in controlling apical membrane traffic (e.g., endocytosis and membrane recycling) in polarized colonic epithelial cells. Our results should provide novel insights into the molecular machinery that controls the traffic and functional activity of CFTR CI channels, which are defective or lacking in the most common genetic disorder among Caucasians (i.e, cystic fibrosis).