首页|The role of constitutive PKA-mediated phosphorylation in the regulation of basal I_(Ca) in isolated rat cardiac myocytes
The role of constitutive PKA-mediated phosphorylation in the regulation of basal I_(Ca) in isolated rat cardiac myocytes
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1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca~(2+) current (I_(Ca)) observed in the absence of exogenous β-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 μmol 1~(-1)) increased basal I_(Ca) (P < 0.05), whereas H-89 inhibited I_(Ca) in a concentration-dependent manner with an IC_(50) ～5 μmol 1~(-1). H-89 also inhibited the response to 1.0 μmol 1~(-1) isoprenaline, although relatively high concentrations (30 μmol 1~(-1)) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 μmol 1~(-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 μmol 1~(-1) H-89 or 1 μmol 1~(-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 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.
cardiac myocytescalcium currentprotein kinase AH-89calyculin A
Nicolas Bracken、Moutaz ElKadri、George Hart、Munir Hussain
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Department of Medicine, School of Clinical Sciences, University Clinical Departments, University of Liverpool, Daulby Street, Liverpool L69 3GA
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