ABNORMAL SUBCELLULAR-LOCALIZATION OF MUTATED CFTR PROTEIN IN A CYSTIC-FIBROSIS EPITHELIAL-CELL LINE

The cystic fibrosis gene product, CFTR, is a Cl- channel that possesse s specific binding sites for cytosolic ATP and is activated by cAMP-de pendent protein kinase. Most recently, it was reported that CFTR local izes at the surface apical compartment of normal airway epithelial cel ls, but accumulates in the cytosol of airway cells from CF patients wi th the Delta F508 mutation. In order to explore whether the same diffe rence exists in normal and CF established cell lines that are commonly used in physiological and pharmacological investigations of the CF de fect, eve employed monoclonal antibodies raised against synthetic pept ides corresponding to two different regions of the CFTR protein. One a ntibody (MATG 1061) was generated against amino acids 503-515 Delta 50 8 in the nucleotide binding domain 1, whereas the other (MATG 1031) wa s generated against amino acids 107-117 situated in a putative externa l loop. We used confocal laser scanning microscopy to localize the CFT R protein in T84 (a colonic derived carcinoma), CAPAN-1 (a pancreatic carcinoma), and in CFPAC-1 (a pancreatic carcinoma homozygous for the Delta F508 deletion) cell lines. In permeabilized T84 and CAPAN-1 cell s, immunolabeling with MATG 1061 predominated at the apical domain. By contrast, CFTR staining with MATG 1061 was homogeneously distributed in the cytoplasm of CFPAC-1 cells. In non-permeabilized non-CF cell li nes, MATG 1031 specifically labeled an apical membrane surface epitope . No such labeling was present in CFPAC-1 cells. In Cl-36 efflux exper iments and in patch-clamp experiments using the permeabilized patch co nfiguration, increased Cl- permeability in response to cAMP stimulatio n was observed in non-CF cell lines but not in CFPAC-1. Our data indic ate a marked difference in CFTR localization between non-CF epithelial cell lines and CFPAC-1 cells homozygous for the Delta F508 deletion. Mislocation of Delta F508CFTR in CFPAC-1 is likely responsible for the functional defective response to increased cAMP.