The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride selective ion channel, regulated by phosphorylation and nucleotide binding. It is predominantly located in the apical membrane of secretory and reabsorptive epithelia, aiding transcellular fluid transport. Immunological and electrophysiological studies have indicated enrichment at the apical membrane, but whether this is the only location where CFTR functions is currently unknown. In cystic fibrosis (CF) patients, the ion and fluid transport is profoundly affected by the incorrect location or function of CFTR. The major CF mutation (CFTRdeltaF508) found in 68% of CF alleles causes the arrest of the protein in the endoplamic reticulum. Other mutant CFTR, including those with amino acid alterations found in pancreatic sufficient (PS) patients appear to be arrested at subsequent stages along the exocytic pathway. Since pancreatic function is a clinical measure which correlates with genotype, we anticipate that pancreatic sufficient mutants will provide insight into the intracellular function, interactions and specific sorting events that direct the delivery of CFTR to the apical surface. The objectives of our proposal are 1) to examine the location and function of CFTR that contain the missense mutations identified in pancreatic sufficient patients, 2) to determine how CFTR is targeted to the apical membrane of epithelia and 3) to examine the nature of the interactions that occur between CFTR and other proteins at, or enroute to the apical membrane.
These aims are directed toward understanding how molecular defects in CFTR cause CF. Specifically, CFTR variants containing the missense mutations found in CF patients will be generated by site directed mutagenesis and expressed in epithelial cells. Cell fractionation, immunological localization, and pulse chase metabolic labeling experiments will determine the order of trafficking events relative to that of wild type CFTR. Electrophysiological measurements will define the mutant CFTR chloride channel characteristics and discriminate dysfunction from mislocalization effects. Further, the identification of those proteins that may interact with the wild type or mutant proteins via in vitro and in vivo binding will yield insight into CFTR functions. The broadening picture of the phenotypes in CF emphasize the vital role that CFTR plays in several epithelia. The deficiencies in specific tissues as well as general manifestations of disease have implications for designing therapeutic strategies to treat CF. A complete understanding of the events of trafficking and their relation to the function of CFTR will be necessary to distinguish between the problems posed by inappropriately located, but active CFTR or those posed by targeted, but dysfunctional CFTR.
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