Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca(2+) current (I(Ca)) by cytosolic free Mg(2+) concentration ([Mg(2+)](i)) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I(Ca), rat cardiac myocytes and tsA201 cells expressing L-type Ca(2+) channels were whole cell voltage-clamped with patch pipettes in which [Mg(2+)] ([Mg(2+)](p)) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca(2+) channels (alpha(1C)/beta(2A)/alpha(2)delta), increasing [Mg(2+)](p) from 0.2 mM to 1.8 mM decreased peak I(Ca) by 76 +/- 4.5% (n = 7). Mg(2+)-dependent modulation of I(Ca) was also observed in cells loaded with ATP-gamma-S. With 0.2 mM [Mg(2+)](p), manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP(2A)) produced large changes in I(Ca) amplitude; however, with 1.8 mM [Mg(2+)](p), these same manipulations had no significant effect on I(Ca). With mutant channels lacking principal PKA phosphorylation sites (alpha(1C/S1928A)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) had only small effects on I(Ca). However, when channel open probability was increased by alpha(1C)-subunit truncation (alpha(1CDelta1905)/beta(2A/S478A/S479A)/alpha(2)delta), increasing [Mg(2+)](p) greatly reduced peak I(Ca). Correspondingly, in myocytes voltage-clamped with pipette PP(2A) to minimize channel phosphorylation, increasing [Mg(2+)](p) produced a much larger reduction in I(Ca) when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg(2+) modulates the extent to which channel phosphorylation regulates I(Ca). This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation.