Since the cloning of the cystic fibrosis (CF) gene, my laboratory has been involved in studies designed to assess function of the protein product, the Cystic Fibrosis Transmembrane Regulator (CFTR). Our study of the purified protein in lipid bilayers provides the most direct evidence that CFTR is capable of function as a phosphorylation dependent chloride (Cl-) channel. In the long term, our goals are to understand the mechanism of action of CFTR and to identify the molecular basis for disease caused by CF mutations. Specifically, we propose to examine the regulation of CFTR by ATP> Recent data have led to the hypothesis that ATP is necessary, not only for phosphorylation-dependent gating but also for an additional gating mechanism requiring ATP hydrolysis. We plan to examine this hypothesis by assessing the regulation of purified, reconstituted CFTR by nucleotides. We will determine defective regulation by ATP underlies disease caused by 'severe' and 'mild' mutations in the putative nucleotide binding folds by assessing activity of purified, reconstituted mutant proteins. Secondly, we plan to examine the capacity for purified CFTR channels to interact with one another. Preliminary patch-clamp data indicate that multiple CFTR channels exhibit cooperatively. Hence, intermolecular interaction may represent another mechanism through which CFTR function may be regulated. This hypothesis will be investigated in studies of purified CFTR and the purified mutant CFTRdeltaF508.
Specific Aim : )A) Role of ATP in regulation of CFTR activity: i) to study regulation of purified CFTR activity by ATP hydrolysis: We will study the Cl- channel activity of purified CFTR and its regulation by ATP, nonhydrolyzable analogues of ATP and ADP. In this and subsequent studies, Cl- channel activity will be assessed using a flux assay of the proteoliposomes and in planar bilayer studies. ii) to study regulation of purified 'R' domain deletion mutant, CFTRdeltaR603-777, by nucleotides. This 'R' domain deletion mutant has lost its' capacity for regulation by phosphorylation. This mutant provides us with the opportunity to study the effects of ATP binding on CFTR channel gating independent of its role in activation by phosphorylation. iii) to study regulation of purified CF mutants by nucleotides. To define the structural basis for regulation by ATP, we will assess the activity of purified, mutant versions of CFTR with lesions localized to the nucleotide binding folds.
Specific Aims B: Interactions between CFTR proteins: i) to determine if purified CFTR functions as a monomer. Liposomes containing single monomers or covalently crosslinked dimers will be physically isolated and tested for Cl- channel function. ii) to determine if purified CFTR exhibits cooperativity. Recent studies suggest that multiple CFTR channels do not behave independently but rather, exhibit cooperativity. In order to determine whether cooperativity is mediated through direct interaction between CFTR molecules, we plan to study kinetic properties of multiple purified CFTR channels in planar lipid bilayers. Our understanding of the mechanism of action of CFTR is essential to defining its role in epithelial fluid transport and the pathogenesis of Cystic Fibrosis.
Showing the most recent 10 out of 30 publications
