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J Biol Chem, Vol. 273, Issue 31, 19573-19578, July 31, 1998

Thermodynamics of Binding of Calcium, Magnesium, and Zinc to the N-Methyl-D-aspartate Receptor Ion Channel Peptidic Inhibitors, Conantokin-G and Conantokin-T

Mary Prorok and Francis J. Castellino

From the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic gamma -carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 °C and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2-3 equivalent Ca2+ sites with an average Kd value of 2800 µM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 µM and another of Kd = 311 µM. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 µM and 1.1 µM), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 µM and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kd values of 428 µM, 10.2 µM, and 0.5 µM, respectively. Again, in the case of con-T, a weak (Kd = 410 µM) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the alpha -helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site.

Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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