PKA holoenzyme is functionally coupled to CFTR by AKAPs

Cystic fibrosis transmembrane regulator (CFTR) isreported to be preferentially regulated by membrane-bound proteinkinase A (PKAII). We tested for close physical and functionalassociation of PKA with CFTR in inside-out membrane patches excisedfrom Calu-3 cells. In the presence of MgATP,8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate(CPT-cAMP) increased the product of CFTR channel number and openprobability (from 0.36 ± 0.12 to 1.23 ± 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. ThusCalu-3 membrane isolated from cells retains PKA holoenzyme that isfunctionally coupled to CFTR. PKAII is anchored at specific subcellularsites by A kinase anchoring proteins (AKAPs). Exposure of excisedpatches to HT-31, a peptide that disrupts the association of PKAII andAKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKAholoenzyme in isolated membrane patches is bound to AKAPs. In wholecell voltage-clamp studies, intracellular dialysis of Calu-3 cells withHT-31 blocked the activation of CFTR by extracellular adenosine. Theseresults suggest that AKAPs mediate PKA compartmentalization with CFTRand are required for activation of CFTR by physiological regulators.