Amino acid substitutions in the pore affect the anomalous mole fraction effect of Ca_V1.2 channels

The anomalous mole fraction effect (AMFE) is an important indicator of ion-ion interactions in the pore of voltage-gated Ca²⁺ channels (VGCCs). The residues at position 1144 that differ in several classes of VGCCs are important to the permeation of the pore. Phe-1144 (F, Ca_V1) was substituted with glycine (G, Ca_V2) and lysine (K, Ca_V3) and the effects of mutation on the voltage and concentration dependency of AMFE were observed. Whole-cell currents were recorded in external solutions with Ca²⁺ and Ba²⁺ at the indicated ratios with a total divalent cation concentration of 2, 10 or 20 mM, at holding potentials from -80 to -20 mV. Results showed the ratio of Ba²⁺ to Ca²⁺ currents determined at 2 mM to be different from that determined under higher concentrations for wild-type channels but this ratio was not different when tail currents were evoked at different potentials. AMFE was greatest at relatively positive potentials (-20 mV) and when the total divalent cation concentrations were kept low (2 mM). AMFE was attenuated for F/G while it was accentuated for F/K compared with wild-type, respectively. The results demonstrated that glycine and lysine substitutions of Phe-1144 affect AMFE through different mechanisms. Additionally, residues at position 1144 were shown to be major determinates of channel permeation of several classes of VGCCs.