Purinoceptor-coupled Cl- channels in mouse heart:: a novel, alternative pathway for CFTR regulation

1. P-2-purinoceptors couple extracellular ATP to the activation of a Cl- current (I-Cl,I- ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of I-Cl,I-ATP activation in mouse heart. 2. Extracellular adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; 100 mu M) activated I-Cl,I-ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATP gamma S while a PRC activator, phorbol 12,13-dibutyrate (PDBu) activated a current with identical properties to I-Cl,I-ATP. Maximal activation of I-Cl,I-ATP by ATP gamma S or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl- channels. 3. Isoprenaline increased I-Cl,I-ATP pre-activated by ATP gamma S or PDBu, while isoprenaline or forskolin alone failed to activate any Cl- current in these myocytes. Adenosine 3',5'-cyclic monophosphothionate, a PKA inhibitor, prevented ATP gamma S or PDBu activation of I-Cl,I-ATP. Thus, I-Cl,I-ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. 4. Glibenclamide (50 mu M) significantly blocked I-Cl,I-ATP activated by ATP gamma S or by the CFTR channel activator, levamisole. 5. The slope conductance of the unitary I-Cl,I-ATP in cell-attached patches was 11.8 +/- 0.3 pS, resembling the known properties of CFTR Cl- channels in cardiac myocytes. 6. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 7. We conclude that I-Cl,I-ATP in mouse heart is clue to activation of CFTR Cl- channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.