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

1 Pharmacological inhibitors of protein kinase A(PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca2+ current (I-Ca) observed in the absence of exogenous beta-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I-Ca in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 mu mol l(-1)) increased basal I-Ca (P < 0.05), whereas H-89 inhibited I-Ca in a concentration-dependent manner with an IC50 similar to 5 mu mol l(-1). H-89 also inhibited the response to 1.0 mu mol l(-1) isoprenaline, although relatively high concentrations (30 mu mol l(-1)) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 mu mol l(-1) H-89 on time-dependent recovery of I-Ca from voltage-dependent inactivation as well as the steady-state gating of I-Ca. T-0.5 (time for I-Ca to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P < 0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of I-Ca were unaffected by 10 mu mol l(-1) H-89 or 1 mu mol l(-1) calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V-0.5 for activation and inactivation became more negative. 5 Data show that basal I-Ca is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied beta-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to beta-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.