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

Authors


Abstract

  • 1P2-purinoceptors couple extracellular ATP to the activation of a Cl current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart.
  • 2Extracellular adenosine-5′-O-(3-thiotriphosphate) (ATPγS; 100 μM) activated ICl,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 ICl,ATP. Maximal activation of ICl,ATP by ATPγS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl channels.
  • 3Isoprenaline increased ICl,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 ICl,ATP. Thus, ICl,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.
  • 4Glibenclamide (50 μM) significantly blocked ICl,ATP activated by ATPγS or by the CFTR channel activator, levamisole.
  • 5The slope conductance of the unitary ICl,ATP in cell-attached patches was 11·8 ± 0·3 pS, resembling the known properties of CFTR Cl channels in cardiac myocytes.
  • 6The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart.
  • 7We conclude that ICl,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.

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