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

Abstract

1. P2-purinoceptors couple extracellular ATP to the activation of a Cl- current (I(Cl,ATP)) in heart. We studied the molecular mechanism and intracellular signalling pathways of I(Cl,ATP) activation in mouse heart. 2. Extracellular adenosine-5'-O-(3-thiotriphosphate) (ATPγS: 100 μM) activated I(Cl,ATP) in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPγS while a PKC activator, phorbol 12,13-dibutyrate (PDBu) activated a current with identical properties to I(Cl,ATP). Maximal activation of I(Cl,ATP) by ATPγS 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,ATP) pre-activated by ATPγS 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 ATPγS or PDBu activation of I(Cl,ATP). Thus, I(Cl,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 μM) significantly blocked I(Cl,ATP) activated by ATPγS or by the CFTR channel activator, levamisole. 5. The slope conductance of the unitary I(Cl,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,ATP) in mouse heart is due to activation of CFTR Cl- channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.