Molecular Mechanisms of CFTR Function

描述（由申请人提供）：囊性纤维化跨膜传导调节因子（CFTR）是ABC转运蛋白超家族的成员，是囊性纤维化患者基因突变的产物。人类基因组中48个ABC蛋白基因中的许多基因（包括CFTR）的突变或过表达与人类疾病有关。因此，了解这些分子的结构和功能具有重要的基础和实际意义。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。它不是利用ATP在其两个核苷酸结合结构域（NBD）处的结合和水解来介导有机溶质的转运，而是利用ATP作为可水解配体来调节其氯离子通道孔的门控。该功能对于维持上皮表面的盐和流体稳态至关重要，特别是在胃肠道和呼吸道中。该项目的目的是测试CFTR是配体门控通道的假设，其中配体水解提供了门控循环的有效可逆性。ATP以高亲和力结合，并在NBD 1处被封闭，以促进NBD 2处的MgATP结合，从而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和门控终止时松弛。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。两个非等效NBD在核苷酸结合/水解和通道门控中的特定作用，2.）蛋白激酶A的磷酸化如何使核苷酸调控门控而不影响其与NBD的相互作用，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。将通过光亲和标记测定ATP结合和水解。纯化和重构的蛋白质将用于ATP酶测定，用于已经实现的2D晶体结构测定的继续，以及用于3D结晶试验。

DESCRIPTION (provided by applicant): 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.