Cystic fibrosis (CF) is an autosomal recessive disease affecting Cl- secretion in a number of tissues such as airway, pancreas and intestine. Since the recent discovery of the gene defective in CF, studies of the expressed protein (designated CFTR) have suggested that the normal gene product is a cAMP-regulated Cl- channel, or a regulator of Cl- channels. Differences between the regulation of Cl- secretion in intestinal cells and airway cells encourages the development of intestinal model systems to examine how changes in CFTR level and CFTR structure affect Cl- channel regulation. The long term objective in this work is to establish an intestinal model system with which to examine the functions of CFTR isoforms made in normal individuals, and mutant CFTR proteins made by CF patients. We propose to develop a HT29-Cl model system in which homologous recombination has been used to specifically disrupt the CFTR gene. HT29-Cl (a human colon carcinoma cell line) is selected because these cells demonstrate transepithelial Cl- secretion, high levels of CFTR mRNA, and a single Cl- conductance regulated by both cAMP and Ca2+. Mutant HT29-Cl with disrupted CFTR genes will be a valuable model as cells having all the machinery for regulated Cl- secretion except CFTR. These mutant cells will be transfected with CFTR cDNAs and analyzed for changes in Cl- channel regulation. Experiments will use SPQ (a fluorescent dye used to measure Cl- activity) to screen for CFTR-dependent changes in Cl- channel activation in polarized cells. Cl- currents will then be characterized in detail using the whole cell patch clamp technique. Transfection with normal CFTR cDNAs will be used to define the relationship between CFTR gene dosage and Cl- secretion. HT29-Cl mutants without functional CFTR genes will be transfected with mutant CFTR cDNAs that contain mutations in the first nucleotide binding fold (NBF1). Missense mutations will be designed 1) to disrupt a putative protein kinase phosphorylation site in NBF1, or 2) mimic CF mutations in NBF1 that have been associated with either """"""""mild"""""""" or """"""""severe"""""""" disease. A third transfected CFTR cDNA will mimic a CFTR mRNA population found in normal individuals (9- mRNA), caused by alternative RNA splicing of exon 9 from full length CFTR mRNA. Additional experiments using both the normal HT29-Cl and HT29-18 cell lines will test whether translation of endogenous 9- mRNA results in measurable amounts of a 9- CFTR protein isoform, define the membrane localization of CFTR isoforms, and determine the general mechanisms regulating CFTR synthesis in HT29 cells.
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