Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes

The effects of changing cytosolic [Mg²⁺] ([Mg²⁺]_i) on L-type Ca²⁺ currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg²⁺] and [Ca²⁺]. To control [Mg²⁺]_i and cytosolic [Ca²⁺] ([Ca²⁺i]_i), the pipette solution included 30 mm citrate and 10 mM ATP along with 5 mM EGTA (slow Ca²⁺ buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca²⁺ buffer). With pipette [Ca²⁺] ([Ca²⁺]_p) set at 100 mM using a slow Ca²⁺ buffer and pipette [Mg²⁺] ([Mg²⁺]_p) set at 0.2 mM, peak L-type Ca²⁺ current density (I_Ca) was 17.0 ± 2.2 pA pF^-1. Under the same conditions, but with [Mg²⁺]_p set to 1.8 mM, I_Ca was 5.6 ± 1.0 pA pF^-1, a 64 ± 2.8% decrease in amplitude. This decrease in I_Ca was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg²⁺]_p-dependent decrease in I_Ca was not significantly different when myocytes were preincubated with 10 μM forskolin and 300 μM 3-isobutyl-1-methylxanthine and voltage-clamped with pipettes containing 50 μM okadaic acid, to maximize Ca²⁺ channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I_Ca decreased only 25 ± 3.4% on changing [Mg²⁺]_p from 0.2 to 1.8 mM. In the presence of 0.2 mM [Mg²⁺]_p, changingchannel phosphorylation conditions altered I_Ca over a 4-fold range; however, with 1.8 mM [Mg²⁺]_p, these same manoeuvres had a much smaller effect on I_Ca. These data suggest that [Mg²⁺]_i can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca²⁺]_p to 1, 100 or 300 nM also showed that the [Mg²⁺]_p-induced reduction of I_Ca was smaller at the lowest [Ca²⁺]_p, irrespective of channel phosphorylation conditions. This interaction between [Ca²⁺]_i and [Mg²⁺]_i to modulate I_Ca was not significantly affected by ryanodine, fast Ca²⁺ buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg²⁺]_i modulates I_Ca by counteracting the effects of Ca²⁺ channel phosphorylation and by an unknown [Ca²⁺]_i-dependent mechanism. The magnitude of these effects suggests that changes in [Mg²⁺]_i could be critical in regulating L-type channel gating.