Mechanisms, Structure, and Regulation of CFTR's NBDs

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DESCRIPTION: CFTR (cystic fibrosis transmembrane conductance regulator), encoded by the gene mutated in CF, is an ATP binding cassette (ABC) protein that forms a Cl- ion channel. Opening and closing of the channel pore are regulated by binding and hydrolysis of ATP at CFTR's two nucleotide binding domains (NBDs) which, in turn, are controlled by cAMP-dependent protein kinase-mediated phosphorylation, and specific phosphatase-mediated dephosphorylation, of particular serine residues concentrated in CFTR's regulatory (R) domain. The goal of the proposed research is to understand, in molecular detail, the structure and mechanisms of function of the NBDs, the interactions between them, and the mechanisms by which they are regulated. Understanding the precise mechanisms that control opening and closing of CFTR Cl- channels might permit eventual pharmacological rescue in CF patients of diseased cells with inadequate ion flow due to expression of mutant CFTR channels; this includes those mutants that fail to reach the cell surface in sufficient numbers, those that have a diminished single-channel conductance, and those that are not open for a large enough fraction of the time. The two specific aims of the project remain unchanged. The first addresses the questions: What do the NBDs look like, how do they function, and what are the mechanisms of interactions between them? The working hypothesis is that the two NBDs are similar, in that they are both capable of binding and hydrolyzing ATP, but they differ in structure, function and mechanism - one becoming "active" only after binding, and likely hydrolysis, of a hydrolyzable nucleoside triphosphate, while nucleotide binding appears sufficient to activate the other. The second aim addresses the questions: How does phosphorylation (and at which site or sites) permit channel opening? How does phosphorylation of an additional labile site (or sites) promote stabilization of the channel open state? Which is that site (or sites)? Wild type and mutant CFTR channels will be expressed in oocytes and mammalian cells, and their structure and function will be analyzed using electrophysiological, biochemical, molecular biological, and biophysical methods (including molecular modeling and, hopefully, eventually crystallography of the key cytoplasmic domains). Rates of opening and closing of single CFTR channels bearing specific mutations in those domains (selected by exploiting models of their structure, and crystal structures of related molecules) will be measured to probe their normal catalytic functions and their cooperative interactions.