Molecular Mechanisms of CFTR Function

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DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in vertebrate epithelial salt and fluid homeostasis and its absence or dysfunction results in cystic fibrosis in humans. In this project we have characterized CFTR single channel gating kinetics, its ability to bind and hydrolyze ATP, and its control by the phosphorylation state of its unique R domain. Findings thus far are consistent with a model in which monomeric CFTR acts as a hydrolysable-ligand gated channel in which there is phosphorylation regulated allosteric coupling between ATP binding/hydrolysis and channel gating. We have found recently that phosphorylation rather than influencing ATP hydrolysis, promotes release of unhydrolysed ATP from NBD1 and also increases the radius of gyration of the largely unstructured R domain which in turn alters the conformation of membrane spanning domains. We are the only group to have purified, crystallized, and determined a low resolution structure of the complete protein by electron crystallography. We have also generated a high resolution model computationally which satisfies a body of published experimental data and reveals domain interactions that we have confirmed by cysteine cross-linking and binding experiments. Domain-swapping interactions have been defined between cytoplasmic and membrane domains in opposite halves of the molecule which are crucial to both its assembly and function. One of these domain-swapping interactions is mediated by the aromatic side chain of phenylalanine residue 508, deleted in most CF patients, which we showed independently is directly involved in channel gating. Our major objectives now are to further elucidate the roles of wild-type CFTR's multiple domains and the interactions between them in its normal function and then to determine how these are altered by the major cystic fibrosis causing mutation, ?F508. The first broad aim will address four significant unresolved issues. The first asks whether unhydrolysed ATP disengagement from the degenerate signature motif of NBD2 and its phosphorylation stimulated dissociation from NBD1 contribute to the opening of the interface between the NBDs and the closing of the channel. Second, we will determine the role of each of the six "transmission interfaces" between the NBDs and MSDs including those that mediate the domain-swapping or intertwining between opposite sides of the molecule. Third, changes in inter-helical relationships in the membrane spanning domains in response to channel activating stimuli will be mapped and their contribution to the ion pore identified. Fourth, the influences of the NBDs and phosphorylation controlled R domain on each other during CFTR function will be determined. Higher resolution 3D structures of different functional states will be determined by electron crystallography in conjunction with these biochemical studies. In the second principal objective motivated by our localization of Phe508 in the 3D structure we will determine the impact of its absence on the structure and function of the rest of the protein in order to facilitate the development of new therapeutic strategies. PUBLIC HEALTH RELEVANCE: CFTR is a unique ion channel employing a modified active transporter structural architecture which when mutated results in cystic fibrosis in humans. The phosphorylation regulated channel provides a rate limiting step in ion and fluid movement across epithelial surfaces in virtually all terrestrial and marine vertebrates. Thus, knowledge of its 3D structure and dynamics during the performance of this function is of both fundamental and practical importance to understanding normal human health and disease.