EFFECTS OF MUTATIONS IN CAMP-DEPENDENT PROTEIN-KINASE ON CHLORIDE EFFLUX IN CACO-2 HUMAN COLONIC-CARCINOMA CELLS

In order to evaluate the importance of cAMP and cAMP-dependent protein kinase (cAMPdPK) in the regulation of chloride efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, Caco-2, human colonic carcinoma cells were transfected with an express ion vector encoding a mutant form of regulatory subunit of cAMPdPK und er control of the mouse metallothionein 1 promoter. Four stable transf ormants were isolated that expressed the mutant subunit in a Zn2+-indu cible manner and exhibited Zn2+-inducible inhibition of cAMPdPK activi ty. The parental and transformed Caco-2 cells were examined for their abilities to regulate chloride efflux in response to various secretago gues using a radioactive iodide-efflux assay. In the transformants, in duction of the protein kinase mutation with ZnSO4 markedly decreased c hloride efflux in response to forskolin, the 8-(4-chlorophenylthio) an alog of cAMP, vasoactive intestinal polypeptide, prostaglandin E2 and isoproterenol, whereas Zn2(+)-treated parental cells remained responsi ve to these secretagogues. Treatment with carbachol, calcium ionophore s or phorbol ester did not acutely affect chloride efflux. Together, t hese studies indicate that cAMP and cAMPdPK are essential components o f secretagogue-regulated chloride channel activity in the Caco-2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK m utation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intrace llularly, reversed the inhibition. These latter results demonstrate th at the CFTR chloride channels in the protein kinase-defective transfor mants are normal and that the protein kinase mutation specifically aff ects their regulation, presumably by direct phosphorylation. (C) 1995 Wiley-Liss, Inc.