Molecular Mechanisms of CFTR Function

囊性纤维化跨膜传导调节因子（CFTR）是ABC转运蛋白的一员， 超家族，是囊性纤维化患者基因突变的产物。突变或过度表达 包括CFTR在内的人类基因组中48个ABC蛋白基因中的许多与人类疾病有关。 因此了解这些分子的结构和功能具有重要的理论和实际意义 重要性CFTR在ABC蛋白中是新颖的，因为它是离子通道而不是转运蛋白。相反 利用ATP在其两个核苷酸结合结构域（NBD）的结合和水解， 一种有机溶质的矢量运输，它利用ATP作为可水解的配体来调节其 氯离子通道孔这种功能对于维持上皮细胞的盐和液体稳态至关重要。 表面，特别是在胃肠道和呼吸道。这个项目的目的是检验假设 CFTR是配体门控通道，其中配体水解提供门控的有效可逆性 周期ATP以高亲和力结合，并在NBD 1处被封闭，以促进MgATP在NBD 2处结合， 扰乱闭合状态构象并启动门控转换。这种熵结构 重排在水解时松弛，门控终止。水解产物的解离允许返回 到初始构象状态。为了检验这一假设，三个具体目标将决定：1。具体 两种非等效NBD在核苷酸结合/水解和通道门控中的作用，2.）如何 蛋白激酶A的磷酸化使得核苷酸调控门控而不影响其功能。 与NBD的互动，3.）核苷酸和PKA对CFTR三维结构的影响。 单通道门控的测量将在平面脂质双层中进行。ATP结合和水解将 通过光亲和标记进行测定。纯化和重构的蛋白质将用于ATP酶测定， 用于继续已经实现的2D晶体结构测定，以及用于3D结晶 审判 城市，州）

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is the product of the gene mutated in patients with cystic fibrosis. Mutations or overexpression of many of the 48 ABC protein genes in the human genome including CFTR are involved in human disease. Hence understanding the structure and function of these molecules is of both fundamental and practical importance. CFTR is novel among ABC proteins in that it is an ion channel rather than a transporter. Instead of harnessing the binding and hydrolysis of ATP at its two nucleotide-binding domains (NBDs) to the vectorial transport of an organic solute, it utilizes ATP as a hydrolysable ligand to regulate the gating of its chloride channel pore. This function is crucial to the maintenance of salt and fluid homeostasis at epithelial surfaces, especially in the GI and respiratory tracts. The objective of this project is to test the hypothesis that CFTR is a ligand-gated channel where ligand hydrolysis provides efficient reversibility of the gating cycle. ATP binds with high affinity and is occluded at NBD1 to promote MgATP binding at NBD2 that perturbs the closed state conformation and initiates gating transitions. This entropic structural rearrangement relaxes on hydrolysis and gating terminates. Dissociation of hydrolysis products allow return to the initial conformational state. To test this hypothesis, three specific aims will determine: 1.) the specific roles of the two non-equivalent NBDs in nucleotide binding/hydrolysis and channel gating, 2.) how phosphorylation by protein kinase A enables nucleotide regulation of gating without influencing its interactions with the NBDs, 3.) the influence of nucleotides and PKA on the 3D structure of CFTR. Measurements of single channel gating will be made in planar lipid bilayers. ATP binding and hydrolysis will be assayed by photoaffinity labeling. The purified and reconstituted protein will be used for ATPase assays, for continuation of 2D crystal structure determination which has been achieved, and for 3D crystallization trials. city, state)