EXPRESSION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN A MODEL EPITHELIUM

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- ch annel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)dependen t phosphorylation and by intracellular nucleotides. The function of CF TR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to dev elop a system to study the function of recombinant CFTR expressed in a n epithelium. We chose Fischer rat thyroid (FRT) epithelia for two rea sons. First, when grown on permeable filter supports, FRT cells form p olarized epithelia with a high transepithelial resistance. Second, the y have no endogenous cAMP-regulated Cl- channels in their apical membr ane. We expressed CFTR in FRT epithelia either transiently, using reco mbinant vaccinia virus, or stably, using a retrovirus. To measure apic al membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CF TR containing the Delta F508 mutation (vTF-Delta F508). These Cl- curr ents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR. However, CFTR was al so expressed in the basolateral membrane: when we permeabilized the ap ical membrane in the presence of a transepithelial Cl- concentration g radient, we observed cAMP-activated Cl- currents in the basolateral me mbrane. Immunocytochemistry confirmed that CFTR was present in both th e apical and basolateral membranes of infected FRT epithelia. This was not a consequence of overexpression, since basolateral cAMP-activated Cl- currents were observed even at low levels of expression of CFTR. Thus the expression of recombinant CFTR in FRT epithelia provides a po werful and convenient system to study the function of CFTR. This metho d may also prove useful when examining the function of other ion chann els.