Cooperative regulation of Ca(v)1.2 channels by intracellular Mg(2+), the proximal C-terminal EF-hand, and the distal C-terminal domain

L-type Ca(2+) currents conducted by Ca(v)1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg(2+) (Mg(i)) inhibits the ionic current of Ca(v)1.2 channels. Because Mg(i) is altered in ischemia and heart failure, its regulation of Ca(v)1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg(i) on voltage-dependent inactivation (VDI) of Ca(v)1.2 channels using Na(+) as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca(2+)-dependent inactivation process. We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion. The increased rate and extent of VDI caused by increased Mg(i) were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg(i) to the EF-hand (K(D) ≈ 0.9 mM) near the resting level of Mg(i) in ventricular myocytes. VDI was more rapid for L-type Ca(2+) currents in ventricular myocytes than for Ca(v)1.2 channels in transfected cells. Coexpression of Ca(v)β(2b) subunits and formation of an autoinhibitory complex of truncated Ca(v)1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca(v)1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.