EPITOPE TAGGING PERMITS CELL-SURFACE DETECTION OF FUNCTIONAL CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a ph osphorylation-activated Cl channel responsible for adenosine 3',5'-cyc lic monophosphate (cAMP)-induced Cl secretion across the apical membra nes of epithelial cells. To optimize its detection for membrane locali zation studies, we tagged CFTR with epitope sequences at the carboxy t erminus or in the fourth external loop. When epitopes were added to th e fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their c AMP-sensitive Cl permeability was assayed using the halide-sensitive f luorophore SPQ. CFTRs containing the M2 epitope showed halide permeabi lity responses to cAMP, whereas cells expressing CFTR with the hemaggl utinin (HA) tag showed little or no cAMP response. Xenopus oocytes exp ressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas MB-tagged c onstructs retaining the glycosylation sites responded like wild-type C FTR. External MB-tagged CFTR was detected in the surface membrane of n onpermeabilized cells. The surface expression of the mutant MB-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mel. Cell. Biol. 11: 3886-3893, 1991). M2-9O1/CFTR is a useful reporter fo r the trafficking of wild-type and mutant CFTRs to the cell surface.