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Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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 ICa, the goal of this study was to identify the counteracting kinase and phosphatase that determine ‘basal’ICa in isolated mouse ventricular myocytes. Whole-cell voltage-clamp studies, with filling solutions containing 10 mM EGTA, revealed that calyculin A (100 nM) increased ICa at test potentials between –42 and +49mV (44% at 0mV) from a holding potential of –80mV. It also shifted the V0.5 (membrane potential at half-maximal) of both activation (from –17 to –25mV) and steady-state inactivation (from –32 to –37mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nM), was without effect on ICa when added after calyculin A. However, by itself, staurosporine decreased ICa throughout the voltage range examined (50% at 0mV) 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 µM in the pipette) and H89 (1 µM) failed to reduce basal ICa or to block the calyculin A-evoked increase in ICa. 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 µM) and Gö 6976 (200 nM) decreased steady-state ICa 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 ICa. However, PP1 is important, as dialysis with 2 µM okadaic acid or 500 nM inhibitor-2 mimicked the increases in ICa 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 ICa.
(Received 19 December 2003;
accepted after revision 21 January 2004;
first published online 23 January 2004)
Corresponding author W. H. duBell: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Email: wdubell{at}som.umaryland.edu
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