Regulation of Cl-Transport in T84 Cell Clones Expressing a Mutant Regulatory Subunit of cAMP-Dependent Protein Kinase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 87; no. 22; pp. 8975 - 8979
• Main Authors: Rogers, Kimberly V., Goldman, Phyllis S., Frizzell, Raymond A., McKnight, G. Stanley
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.11.1990; National Acad Sciences
• Subjects: Biological and medical sciences; Biological Transport; Calcimycin - pharmacology; Calcium - physiology; Cell culture techniques; Cell Line; Cell lines; Cell physiology; Chlorides - metabolism; Cloning, Molecular; Cyclic AMP - physiology; Cystic fibrosis; Drug regulation; Epithelial cells; Epithelium - metabolism; Fundamental and applied biological sciences. Psychology; Hormonal regulation; Hormones; Humans; In Vitro Techniques; Ion channels; Ionomycin - pharmacology; Membrane and intracellular transports; Molecular and cellular biology; Phosphorylation; Protein Kinases - physiology; Receptors, Cyclic AMP - physiology; Secretion; Transfection; Regulation(control); Cell line; Enzyme; Protein kinase; Membrane transport; Second messenger
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.22.8975

Cl-channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+second-messengers systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl-channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+inophores retain their ability to activate the Cl-channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2+-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2+-dependent Cl-channel activation in isolation from interactions with the cAMP second-messenger pathway.