Amino acid substitutions in the pore of the Ca_V1.2 calcium channel reduce barium currents without affecting calcium currents

Ba²⁺ currents through Ca_V1.2 Ca²⁺ channels are typically twice as large as Ca²⁺ currents. Replacing Phe-1144 in the pore-loop of domain III with glycine and lysine, and Tyr-1152 with lysine, reduces whole-cell G_Ba/G_Ca from 2.2 (wild-type) to 0.95, 1.21, and 0.90, respectively. Whole-cell and single-channel measurements indicate that reductions in G_Ba/G_Ca result specifically from a decrease in Ba²⁺ conductance and not changes in V_h or P_O. Half-maximal block of I_Li is increased by 3.2-, 3.8-, and 1.6-fold in Ca²⁺, and 3.8-, 4.2-, and 1.8-fold in Ba ²⁺ for F1144G, Y1152K, and F1144K, respectively. High affinity interactions of individual divalent cations to the pore are not important for determining G_Ba/G_Ca, because the fold increases in IC ₅₀ values for Ba₂₊ and Ca²⁺ are similar. On the contrary, conductance-concentration curves indicate that G_Ba/G _Ca is reduced because the interactions of multiple Ba²⁺ ions in the mutant pores are altered. The complexity of these interactions is exemplified by the anomalous mole fraction effect, which is flattened for F1144G and FY/GK but accentuated for F1144K. In summary, the physicochemical properties of the amino acid residues at positions 1144 and 1152 are crucial to the pore's ability to distinguish between multiple Ba²⁺ ions and Ca²⁺ ions.