THERMODYNAMICS OF BINDING OF CALCIUM, MAGNESIUM, AND ZINC TO THE N-METHYL-D-ASPARTATE RECEPTOR-ION CHANNEL PEPTIDIC INHIBITORS, CONANTOKIN-G AND CONANTOKIN-T

The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Z n2+, to the synthetic gamma-carboxyglutamic acid-containing neuroactiv e peptides, conantokin-G (con-G) and conantokin-T (con-T), have been d etermined by isothermal titration calorimetry (ITC) at 25 degrees C an d 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 mu M. With Mg2+, the ligand, two separate exothermic sites are o btained by ITC, one of K-d = 46 mu M and another of K-d = 311 mu M. Mu ch tighter binding of Zn2+ is observed for these latter two sites (K-d values = 0.2 mu M and 1.1 mu M), and a third considerably weaker bind ing site is observed, characterized by a K-d value of 286 mu M and an endothermic enthalpy of binding, con-T possesses a single exothermic t ight binding site for Ca2+, Mg2+, and Zn2+, with K-d values of 428 mu M, 10.2 mu M, and 0.5 mu M, respectively. Again, in the case of con-T, a weak (K-d = 410 mu M) endothermic binding site is observed for Zn2, The binding of these cations to con-G and con-T result in an increas e 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 t hree cations to the peptides are substantially greater than their comp arative 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 th e 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 li kely the most significant metal ion ligands of these peptides in neuro nal cells.