H-89 inhibits transient outward (Ito) and inward rectifier (IK1) potassium currents independently of pka-mediated phosphorylation in isolated rat ventricular myocytes

Abstract

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.