Premium
Effects of mutations in cAMP‐dependent protein kinase on chloride efflux in Caco‐2 human colonic carcinoma cells
Author(s) -
Krolczyk Arkadiusz J.,
Bear Christine E.,
Lai Patrick F. H.,
Schimmer Bernard P.
Publication year - 1995
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041620109
Subject(s) - efflux , caco 2 , protein kinase a , chemistry , kinase , chloride , mutation , biochemistry , cancer research , microbiology and biotechnology , biology , gene , in vitro , organic chemistry
Abstract 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 expression vector encoding a mutant form of regulator subunit of cAMPdPK under control of the mouse metallothionein 1 promoter. Four stable transformants were isolated that expressed the mutant subunit in a Zn 2+ ‐inducible manner and exhibited Zn 2+ ‐inducible inhibition of cAMPdPK activity. The parental and transformed Caco‐2 cells were examined for their abilities to regulate chloride efflux in response to various secretagogues using a radioactive iodide‐efflux assay. In the transformants, induction of the protein kinase mutation with ZnSo 4 markedly decreased chloride efflux in response to forskolin, the 8‐(4‐chlorophenylthio) analog of cAMP, vasoactive intestial polypeptide, prostaglandin E2 and isoproterenol, whereas Zn 2+ ‐treated parental cells remained responsive to these secretagogues. Treatment with carbachol, calcium ionophores or phorbol ester did not acutely affect chloride efflux. Together, these studies indicate that cAMP and cAMPdPK are essential components of secretagogue‐regulated chloride channel activity in the Caco‐2 cell line. In whole cell patch clamp recordings, induction of the cAMPdPK mutation inhibited anionic conductances indicative of the CFTR chloride channel, whereas purified catalytic subunit of cAMPdPK, added intracellularly, reversed the inhibition. These latter results demonstrate that the CFTR chloride channels in the protein kinase‐defective transformants are normal and that the protein kinase mutation specifically affects their regulation, presumably by direct phosphorylation. © 1995 Wiley‐Liss, Inc.