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

The effects of changing cytosolic [Mg 2+ ] ([Mg 2+ ] i ) on l ‐type Ca 2+ currents were investigated in rat cardiac ventricular myocytes voltage‐clamped with patch pipettes containing salt solutions with defined [Mg 2+ ] and [Ca 2+ ]. To control [Mg 2+ ] i and cytosolic [Ca 2+ ] ([Ca 2+ ] i ), the pipette solution included 30 m m citrate and 10 m m ATP along with 5 m m EGTA (slow Ca 2+ buffer) or 15 m m EGTA plus 5 m m BAPTA (fast Ca 2+ buffer). With pipette [Ca 2+ ] ([Ca 2+ ] p ) set at 100 n m using a slow Ca 2+ buffer and pipette [Mg 2+ ] ([Mg 2+ ] p ) set at 0.2 m m , peak l ‐type Ca 2+ current density ( I Ca ) was 17.0 ± 2.2 pA pF −1 . Under the same conditions, but with [Mg 2+ ] p set to 1.8 m m , 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 [Mg 2+ ] p ‐dependent decrease in I Ca was not significantly different when myocytes were preincubated with 10 μ m forskolin and 300 μ m 3‐isobutyl‐1‐methylxanthine and voltage‐clamped with pipettes containing 50 μ m okadaic acid, to maximize Ca 2+ 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 [Mg 2+ ] p from 0.2 to 1.8 m m . In the presence of 0.2 m m [Mg 2+ ] p , changing channel phosphorylation conditions altered I Ca over a 4‐fold range; however, with 1.8 m m [Mg 2+ ] p , these same manoeuvres had a much smaller effect on I Ca . These data suggest that [Mg 2+ ] i can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca 2+ ] p to 1, 100 or 300 n m also showed that the [Mg 2+ ] p ‐induced reduction of I Ca was smaller at the lowest [Ca 2+ ] p , irrespective of channel phosphorylation conditions. This interaction between [Ca 2+ ] i and [Mg 2+ ] i to modulate I Ca was not significantly affected by ryanodine, fast Ca 2+ buffers or inhibitors of calmodulin, calmodulin‐dependent kinase and calcineurin. Thus, physiologically relevant [Mg 2+ ] i modulates I Ca by counteracting the effects of Ca 2+ channel phosphorylation and by an unknown [Ca 2+ ] i ‐dependent mechanism. The magnitude of these effects suggests that changes in [Mg 2+ ] i could be critical in regulating l ‐type channel gating.