The long-range goal of the proposed research is to investigate the sequence of events which leads to the activation of Cl conductance in cells expressing CFTR and to understand how disease-causing mutations alter the sensitivity to activating conditions. Cystic Fibrosis is the most common lethal, recessively inherited disease among caucasians, affecting nearly 1 in 2500 newborns, and the disease is caused by mutations in the gene coding for a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Wild type CFTR has been associated with the expression of a cAMP activated, Cl-selective conductance in a variety of cell types, and until recently it was thought that this Cl conduction was absent in cells expressing disease-causing, mutant CFTRs, particularly deltaF508, the most common mutation associated with severe cystic fibrosis. We have shown, however, that in Xenopus oocytes expression of deltaF508 and other mutant CFTRs is associated with a cAMP activatable Cl conductance which exhibits a markedly reduced sensitivity to an activating stimulus (forskolin + IBMX). Most importantly, the reduction in sensitivity was highly correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. We propose to characterize in detail the activation of Cl conductance in Xenopus oocytes expressing wild type and mutant CFTRs. A quantitative description of the events leading to the activation of chloride conductance should reveal the points at which mutations alter activation.
The specific aims are: 1. To characterize the sequential reaction steps involved in the activation of Cl channels by cAMP in oocytes expressing CFTR. 2. To characterize the conduction properties associated with the expression of wild type CFTR in oocytes. 3. To use specific mutations to evaluate the functional significance of the five putative structural domains of CFTR. The results of these studies could provide a mechanistic basis for the design of a rational drug therapy to ameliorate the symptoms of C.F., which appear to be largely due to insufficient Cl secretion. This proposal has been designed to interact closely with a second R01 submitted by Mitchell Drumm which emphasizes cell-specific expression of CFTR and the identification of therapeutic modalities in different cell types.
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