Effect of Mg_i on VDI of WT Ca_V1.2 and C-terminal EF-hand mutant channels expressed in tsA-201 cells using Na⁺ as charge carrier. Currents were elicited by 1,000-ms steps to 0 mV from an HP of −80 mV. (A) Dependence of VDI of Ca_V1.2(Na) current on Mg_i. n = 8, 12, 19, 15, and 15 for 0.26 0.38, 0.8, 2.4, and 7.2 mM Mg_i, respectively. (B) Plot of r₁₀₀₀ versus log[Mg_i]. Curve represents the best fit of a binding isotherm to the data. The apparent K_d was 0.9 ± 0.1 mM. (C) Effects of Mg_i on VDI of Ca_V1.2 with the coexpression of CaM₁₂₃₄. A calmodulin (CaM) construct containing alanine substitutions for critical aspartate residues that impair Ca²⁺ coordination in the paired EF-hand of both lobes of CaM (CaM₁₂₃₄) was expressed in molar excess relative to the Ca²⁺ channel subunits. n = 3 and 4 for 0.26 and 7.2 mM Mg_i, respectively. (D) Dependence of VDI of C-terminal EF-hand mutant of Ca_V1.2 on Mg_i. Mean ratio of r₁₀₀₀ with 2.4 mM Mg_i to r₁₀₀₀ with 0.26 mM Mg_i (r₁₀₀₀ at 2.4 mM/r₁₀₀₀ at 0.26 mM) for each Ca_V1.2 C-terminal EF-hand mutant. Number of cells at 0.26 and 2.4 mM Mg_i were WT: 9 and 11; D1546A: 7 and 9; D1546N: 6 and 4; D1546S: 6 and 4; D1546R: 5 and 7; and D1546K: 4 and 6.