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 GCl is independent of protein kinase A. We activated G proteins and monitored CFTR GCl in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 muM) stimulated CFTR G(Cl) in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR GCl required Mg2+ 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 mM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 muM); and 3) independent of Ca2+, suggesting that Ca2+-dependent protein kinase C and Ca2+/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 GCl endogenously via a cAMP-independent pathway in this native absorptive epithelium.