Thermodynamics of binding of calcium, magnesium, and zinc to the N- methyl-D-aspartate receptor ion channel peptidic inhibitors, conantokin-G and conantokin-T

Abstract

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic γ-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 K(d) value of 2800 μM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of K(d) = 46 μM and another of K(d) = 311 μM. Much tighter binding of Zn2+ is observed for these latter two sites (K(d) values = 0.2 μM and 1.1 μM), and a third considerably weaker binding site is observed, characterized by a K(d) 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 K(d) values of 428 μM, 10.2 μM, and 0.5 μM, respectively. Again, in the case of con-T, a weak (K(d) = 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 α-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 K(d) 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 K(d) 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.