Functional cystic fibrosis transmembrane conductance regulator tagged withan epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phospho rylation-activated chloride channel apically localized in epithelial cells, In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, delete d at the phenylalanine residue at position 508 (Delta F508), CFTR-Delta F50 8 fails to exit the endoplasmic reticulum; it remains incompletely glycosyl ated and is rapidly degraded. To optimize CFTR detection for membrane local ization studies and biochemical studies, we tagged wild-type and Delta F508 CFTR with the VSV-G epitope at their carboxyterminal ends, We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV -G-tagged human wild-type and Delta F508-CFTR, In CFTR-expressing cells, th e transfected protein is maturated and transported to the apical membrane w here it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescen t dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wildty pe CFTR. In contrast, in cells expressing tagged Delta F508-CFTR or in non- transfected cells, no anion channel activity could be detected after stimul ation by cAMP, In Delta F508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the end oplasmic reticulum, These cell lines reproduce the cellular fate of wild-type and mutated CFTR- Delta F508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-Delta F508 in which cellu lar processing and functional activity of these two proteins are reproduced , Thus the addition of the VSV-G epitope does not impair the localization a nd function of CFTR, and these cell lines can be used to examine CFTR funct ion in vitro.