Protein phosphatase 1 and an opposing protein kinase regulate steady-state L-type Ca2+ current in mouse cardiac myocytes

Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca2+ current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L-type I-Ca, the goal of this study was to identify the counteracting kinase and phosphatase that determine 'basal' I-Ca in isolated mouse ventricular myocytes. Whole-cell voltage-damp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I-Ca attest potentials between -42 and +49 mV (44% at 0 mV)from a holding potential of -80 mV. It also shifted the V-0.5 (membrane potential at half-maximal) of both activation (from -17 to -25 mV) and steady-state inactivation (from -32 to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on I-Ca when added after calyculin A. However, by itself, staurosporine decreased I-Ca throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs; (100 mum in the pipette) and H89 (1 mum) failed to reduce basal I-Ca or to block the calyculin A-evoked increase in I-Ca. Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba2+ was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31-8220 (1 mum) and Go 6976 (200 nm) decreased steady-state I-Ca and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10-100 nm okadaic acid or 500 nm fostriecin failed to increase I-Ca. However, PP1 is important, as dialysis with 2 mum okadaic acid or 500 nm inhibitor-2 mimicked the increases in I-Ca seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I-Ca.