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

摘要

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), 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 nm using a slow Ca²⁺ buffer and pipette [Mg²⁺] ([Mg²⁺]p) set at 0.2 mm, peak l-type Ca²⁺ current density (ICa) was 17.0 ± 2.2 pA pF⁻¹. Under the same conditions, but with [Mg²⁺]p set to 1.8 mm, ICa was 5.6 ± 1.0 pA pF⁻¹, a 64 ± 2.8% decrease in amplitude. This decrease in ICa was accompanied by an acceleration and a –8 mV shift in the voltage dependence of current inactivation. The [Mg²⁺]p-dependent decrease in ICa 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, ICa 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, changing channel phosphorylation conditions altered ICa 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 n class="small-caps">m 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 class="small-caps">l-type channel gating.