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

作者：William H. duBell, Terry B. Rogers · 发表于：The Journal of Physiology · 年份：2004 · DOI：10.1113/jphysiol.2003.059329 · 被引用次数：47 · 研究领域：Ion channel regulation and function、Cardiac electrophysiology and arrhythmias、Protein Kinase Regulation and GTPase Signaling

Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca(2+) 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-clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I(Ca) at test 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 microm in the pipette) and H89 (1 microm) 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 Ba(2+) was used as the charge carrier...