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Amino Acid Substitutions in the Pore of the Ca_V1.2 Calcium Channel Reduce Barium Currents without Affecting Calcium Currents

Xianming Wang ^*, Tudor A. Ponoran , Randall L. Rasmusson , David S. Ragsdale  and Blaise Z. Peterson ^*

^* Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; Montreal Neurological Institute, McGill University, Montreal, Quebec H3A2B4, Canada; and The Department of Physiology and Biophysics, University at Buffalo, The State University of New York, School of Medicine and Biomedical Sciences, Buffalo, New York 14214

Correspondence: Address reprint requests to B. Z. Peterson, E-mail: bpeterson{at}psu.edu.

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 Ba²⁺ 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 IC₅₀ 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.

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