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

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)dependent phosphorylation and by intracellular nucleotides. The function of CFTR, 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 develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical 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 CFTR containing the Delta F508 mutation (vTF-Delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR. However, CFTR was also expressed in the basolateral membrane: when we permeabilized the apical membrane in the presence of a transepithelial Cl- concentration gradient, we observed cAMP-activated Cl- currents in the basolateral membrane. Immunocytochemistry confirmed that CFTR was present in both the 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 powerful and convenient system to study the function of CFTR. This method may also prove useful when examining the function of other ion channels.