Modulation of Ca_V1.2 Channels by Mg²⁺ Acting at an EF-hand Motif in the COOH-terminal Domain

Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mg_i) inhibits L-type Ca²⁺ currents through Ca_V1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mg_i acts through the COOH-terminal EF-hand of Ca_V1.2. 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 Mg_i reduced both Ba²⁺ and Ca²⁺ currents conducted by wild type (WT) Ca_V1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT Ca_V1.2 to lower Mg_i (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 Mg_i 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 Mg_i. The effects of Mg_i on peak Ba²⁺ currents were approximately fit by a single binding site model with an apparent K_d of 0.65 mM. The apparent K_d for this effect of Mg_i 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 Mg_i up to 7.2 mM. In contrast to these results, peak Ba²⁺ currents through the K1543D mutant were inhibited by lower concentrations of Mg_i compared with WT, consistent with approximately fourfold reduction in apparent K_d for Mg_i, and inhibition of mutant K1539D by Mg_i was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mg_i 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 Ca_V1.2 channels by Mg_i. Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of Ca_V1.2 channels, and reveal a potentially important role of Mg_i binding to the COOH-terminal EF-hand in regulating Ca²⁺ influx in physiological and pathophysiological states.