PHOSPHORYLATION BY PROTEIN-KINASE-C IS REQUIRED FOR ACUTE ACTIVATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR BY PROTEIN-KINASE-A

Protein kinase A (PKA) stimulates Cl secretion by activating the cysti c fibrosis transmembrane conductance regulator (CFTR), a tightly regul ated Cl- channel in the apical membrane of many secretory epithelia. T he CFTR channel is also modulated by protein kinase C (PKC), but the r egulatory mechanisms are poorly understood, Here we present evidence t hat PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA, When patches were excised from transfected Chinese hamster ovary cell s, CFTR responses to PKA became progressively smaller with time and ev entually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PRC and was reversed by the addition of PKC to channels that had become refractory to PKA, PKC enhanced PKA st imulation of open probability without increasing the number of functio nal channels, Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 mu M) reduced the channel activity that could be elic ited by forskolin in cell-attached patches. Moreover, in whole cell pa tches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PRC inhibitors, although an abr upt, partial activation was observed after a delay of >15 min. Modulat ion by PKC was most pronounced when basal PKC phosphorylation was redu ced by briefly preincubating cells with chelerythrine, Constitutive PK C phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is similar to 60% of that attained during in vitro exposure to both kinases. Differences in basal PI(C activity may contribute to the variable cAMP responsivenes s of CFTR channels in different cell types.