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

Abstract

Previous studies have shown that inhibition of L-type Ca2+current ( ICa) by cytosolic free Mg2+concentration ([Mg2+]i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of ICa, rat cardiac myocytes and tsA201 cells expressing L-type Ca2+channels were whole cell voltage-clamped with patch pipettes in which [Mg2+] ([Mg2+]p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca2+channels (α1C/β2A/α2δ), increasing [Mg2+]pfrom 0.2 mM to 1.8 mM decreased peak ICaby 76 ± 4.5% ( n = 7). Mg2+-dependent modulation of ICawas also observed in cells loaded with ATP-γ-S. With 0.2 mM [Mg2+]p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP2A) produced large changes in ICaamplitude; however, with 1.8 mM [Mg2+]p, these same manipulations had no significant effect on ICa. With mutant channels lacking principal PKA phosphorylation sites (α1C/S1928A/β2A/S478A/S479A/α2δ), increasing [Mg2+]phad only small effects on ICa. However, when channel open probability was increased by α1C-subunit truncation (α1CΔ1905/β2A/S478A/S479A/α2δ), increasing [Mg2+]pgreatly reduced peak ICa. Correspondingly, in myocytes voltage-clamped with pipette PP2Ato minimize channel phosphorylation, increasing [Mg2+]pproduced a much larger reduction in ICawhen channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg2+modulates the extent to which channel phosphorylation regulates ICa. 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.