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.
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