cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct.
It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl(-) conductance (G(Cl)) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G(Cl) is independent of protein kinase A. We activated G proteins and monitored CFTR G(Cl) in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 microM) stimulated CFTR G(Cl) in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR G(Cl) required Mg(2+) and ATP hydrolysis (5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR G(Cl) was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 microM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 microM); and 3) independent of Ca(2+), suggesting that Ca(2+)-dependent protein kinase C and Ca(2+)/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10(-6) M forskolin plus 10(-6) M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR G(Cl) endogenously via a cAMP-independent pathway in this native absorptive epithelium.
Reddy
M M
MM
Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093-0831, USA.
Quinton
P M
PM
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000328:Adult; D002118:Calcium / Q000502:physiology; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D016048:Dideoxyadenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D010766:Phosphorylation; D013545:Sweat Glands / Q000378:metabolism*
D000067956:Adenylyl Cyclase Inhibitors; C505032:CFTR protein, human; D004791:Enzyme Inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017759:9-(tetrahydro-2-furyl)-adenine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016048:Dideoxyadenosine; C021008:2',5'-dideoxyadenosine; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000225:Adenine; D002118:Calcium