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Figure S4. The EEEE locus also shapes the Ca/Ba permeation profile. (A) Nonconserved residue between Ca_V2.3 and Ca_V1.2 (Ca_V2.3 (G₁₃₂₃F)), reported to modify the EEEE locus, decreases ICa/IBa (top) and AMFE (bottom) in a proportion expected from the change in experimentally determined divalent cation binding energy (30) at the EEEE locus. Mutation of the other nonconserved residue between Ca_V2.3 and Ca_V1.2 (Ca_V2.3 (S₁₆₁₁C)) has no effect as suspected from its extra-loci location. On the top are shown the Ca²⁺ channel currents recorded during the usual voltage ramps under different Ba²⁺/Ca²⁺ mole fractions on the bottom, the corresponding current-mole fraction curve for each mutant (Ca_V2.3, square; Ca_V2.3(G₁₃₂₃F), inverted triangle; Ca_V2.3(S₁₆₁₁C),circle). (B, left) Simulated ICa/IBa ratios calculated for decreasing free energies at the internal EEEE locus of the Ca_V2.3 channel. (right) Simulated current-mole fraction curves with the decreasing EEEE ΔG° (bottom) depicted on the left. These curves were labeled Ca_V2.3 and Ca_V2.3(G₁₃₁₁F), after comparison with original traces shown on A, and suspected variations in free energy at EEEE (30) loci due to mutation.