No / Voltage clamp was used to investigate the effects of N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a potent inhibitor of PKA, on transient outward K+ current (Ito) and inward rectifying K+ current (IK1) in rat cardiac muscle. Initial experiments, performed using descending voltage ramps, showed that H-89 inhibited both the outward and inward ramp currents in a concentration-dependent manner at concentrations between 5 and 60 ¿mol l¿1. A similar degree of inhibition was observed when Ito and IK1 were recorded using square wave depolarising and hyperpolarising voltage steps, respectively. The IC50 was 35.8 ¿mol l¿1 for Ito and 27.8 ¿mol l¿1 for IK1 compared to 5.4 ¿mol l¿1 for L-type Ca2+ current (ICa). The Hill coefficients for Ito, IK1 and ICa were ¿1.97, ¿1.60 and ¿1.21, respectively. In addition to inhibiting Ito amplitude, H-89 also accelerated the time to peak and the rate of voltage-dependent inactivation so that the time course of Ito was abbreviated. Paired-pulse protocols were performed to study the effects of H-89 on steady-state activation and inactivation as well as recovery from voltage-dependent inactivation. H-89 produced a concentration-dependent rightward shift in voltage-dependent activation but had no significant effect on steady-state inactivation. Recovery from voltage-dependent inactivation was delayed, although this was only visible at the highest concentration (60 ¿mol l¿1) used. In experiments investigating the effects of elevated cyclic AMP, the ß-adrenergic agonist isoprenaline and the phosphatase inhibitor calyculin A had no major effects on Ito or IK1. Data suggest that the effects of H-89 on K+ currents are more complex than simple inhibition of PKA-mediated phosphorylation.

No / 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 ß-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 ¿mol l¿1) increased basal ICa (P<0.05), whereas H-89 inhibited ICa in a concentration-dependent manner with an IC50 ~5 ¿mol l¿1. H-89 also inhibited the response to 1.0 ¿mol l¿1 isoprenaline, although relatively high concentrations (30 ¿mol l¿1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 ¿mol 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 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of ICa were unaffected by 10 ¿mol l¿1 H-89 or 1 ¿mol 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 ß-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to ß-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.