The role of constitutive PKA‐mediated phosphorylation in the regulation of basal ICa in isolated rat cardiac myocytes

Abstract
1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca2+ current (ICa) observed in the absence of exogenous β-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell ICa in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 μmol l−1) increased basal ICa (PICa in a concentration-dependent manner with an IC50 ∼5 μmol l−1. H-89 also inhibited the response to 1.0 μmol l−1 isoprenaline, although relatively high concentrations (30 μmol l−1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 μmol l−1 H-89 on time-dependent recovery of ICa from voltage-dependent inactivation as well as the steady-state gating of ICa. T0.5 (time for ICa to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (PICa were unaffected by 10 μmol l−1 H-89 or 1 μmol l−1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V0.5 for activation and inactivation became more negative. 5 Data show that basal ICa is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied β-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to β-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes. British Journal of Pharmacology (2006) 148, 1108–1115. doi:10.1038/sj.bjp.0706809

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