Modulation of CaV1.2 Channels by Mg2+ Acting at an EF-hand Motif in the COOH-terminal Domain

Abstract

Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mgi) inhibits L-type Ca2+ currents through CaV1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mgi acts through the COOH-terminal EF-hand of CaV1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg2+ affinity. In whole-cell patch clamp experiments, increased Mgi reduced both Ba2+ and Ca2+ currents conducted by wild type (WT) CaV1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT CaV1.2 to lower Mgi (0.26 mM) increased the amplitudes of Ba2+ currents 2.6 ± 0.4–fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mgi to 2.4 or 7.2 mM reduced current amplitude to 0.5 ± 0.1 and 0.26 ± 0.05 of the control level at 0.8 mM Mgi. The effects of Mgi on peak Ba2+ currents were approximately fit by a single binding site model with an apparent Kd of 0.65 mM. The apparent Kd for this effect of Mgi was shifted ∼3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mgi up to 7.2 mM. In contrast to these results, peak Ba2+ currents through the K1543D mutant were inhibited by lower concentrations of Mgi compared with WT, consistent with approximately fourfold reduction in apparent Kd for Mgi, and inhibition of mutant K1539D by Mgi was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mgi was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of CaV1.2 channels by Mgi. Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of CaV1.2 channels, and reveal a potentially important role of Mgi binding to the COOH-terminal EF-hand in regulating Ca2+ influx in physiological and pathophysiological states.