PKA holoenzyme is functionally coupled to CFTR by AKAPs.
Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from 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 open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.
Huang
P
P
Departments of Medicine and CF/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. Pingbo_Huang@med.unc.edu
Trotter
K
K
Boucher
R C
RC
Milgram
S L
SL
Stutts
M J
MJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002451:Cell Compartmentation / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D018408:Patch-Clamp Techniques; D011506:Proteins / Q000494:pharmacology; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
C505032:CFTR protein, human; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; C400224:Ht 31 protein, synthetic; D011506:Proteins; D018047:Receptors, Purinergic P1; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases