Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF), the most common genetic disease among Caucasians. The CFTR (1,480 amino acids) appears to be an integral membrane protein, predicted to have 12 transmembrane spanning domains, two cytoplasmically-located nucleotide binding folds and a 240 amino acid """"""""regulatory"""""""" (R) domain. Although this protein exhibits homology with several ATP-dependent transporters, recent data suggests that it is intimately involved in chloride channel activity rather than active transport. It is possible that the CFTR possesses an additional, yet undiscovered, activity. The long-term goals of this project are to provide insight into the structure and function of the CFTR and the role of this protein in CF disease pathogenesis. Questions regarding the structure of the CFTR will be addressed by characterizing its membrane topography. These particular experiments will involve the use of specific antibodies to determine the membrane orientation of different regions of the protein, including the putative functional domains (nucleotide binding folds and the R domain). The experiments will be performed on cultured cells expressing recombinant forms of the CFTR. The cellular location of wildtype and mutants of the CFTR will be determined using both the recombinant forms of the protein expressed in culture and the endogenous protein from normal and CF patients. This will ascertain the effect of different CF mutations on CFTR targeting. The function and role of the CFTR in disease pathogenesis will be studied using transgenic mice, made with the CFTR(-/-) mouse. These animals do not express CFTR due to an engineered gene disruption event. Wildtype and mutants of the CFTR will be expressed, as transgenes, in the CFTR(-/- ) mice at different levels by exploring various promoters, as well as by analyzing different founder mice. The transgenic animals will be characterized biochemically, electrophysiologically and clinically. The results of these studies will provide insight into the structure and function of the CFTR and the role that specific mutations play in the course of the disease. Knowledge of the actual physiological abnormality that is responsible for the CF phenotype will aid in the rational design of novel therapeutic strategies.
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