PKA holoenzyme is functionally coupled to CFTR by AKAPs

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferenti ally regulated by membrane-bound protein kinase A (PKAII). We tested for cl ose physical and functional association of PKA with CFTR in inside-out memb rane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlo rophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by P KI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular si tes by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevente d CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membr ane patches is bound to AKAPs. In whole cell voltage-clamp studies, intrace llular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR b y extracellular adenosine. These results suggest that AKAPs mediate PKA com partmentalization with CFTR and are required for activation of CFTR by phys iological regulators.