Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasians, affecting approximately 1 in 2500 newborns and is due to mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). Despite gradual improvements in treatment over the past twenty years, most individuals with CF currently do not survive past age 30, and new approaches to treatment are urgently needed. This proposal will investigate the basic pathophysiologic defect produced by a range of CFTR mutations in epithelial cells, with the long range goal of developing specific pharmacological reagents which will activate the mutant protein product. We have already shown that the most common mutation (deltaF508) and a number of other missense mutations produce CFTR in Xenopus oocytes which can be activated by high levels of IBMX by potentiating protein kinase A (PKA) activity. This observation will now be extended to airway epithelial cell lines in order to determine if mutant forms of CFTR are capable of generating a chloride conductance by elevating PKA activity. The intracellular processing of wild-type and mutant CFTR will be simultaneously studied using a specific antibody and the effects of temperature on processing will be examined. Pharmacologic agents which, when used alone, have only a modest impact on CFTR activity and could work synergistically in mutant activation will be tested in combinations, such as priming of CFTR by PKC phosphorylation followed by activation by PKA. For functional analyses, we will develop an epithelial cell line which is transcriptionally active but does not contain functional CFTR in order to have an epithelial cell type which does not have a background CFTR Cl conductance, nor has a mutant CFTR which could be improperly processed. These experiments should clarify the nature of pathogenesis with regards to the extent processing of mutants is involved compared to qualitative changes in CFTR function, and determine the feasibility of activating the mutants in vivo as a therapeutic strategy for CF.
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