Inhibition of NMDA-induced currents by conantokin-G and conantokin-T in cultured embryonic murine hippocampal neurons

Conantokin-G (con-G) and conantokin-T (con-T) are small (17 and 21 amino acids, respectively) γ-carboxyglutamate (Gla) containing peptides derived from the venoms of marine cone snails that are potent and selective inhibitors of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of con-G and con-T on NMDA-evoked responses were evaluated in mouse primary hippocampal neuronal cultures using the whole-cell patch-clamp technique. Under equilibrium conditions, NMDA-induced currents were inhibited by con-G and con-T (10 nM-100 μM) in a dose-dependent manner while maintaining a holding potential of -70 mV. In the presence of saturating amounts of NMDA (100 μM) and glycine (1 μM), the IC₅₀ values obtained were 487±85 nM for con-G and 1030±130 nM for con-T. NMDA (10 μM-1 mM) dose-response curves produced in the presence of con-G or con-T (1 or 3 μM) resulted in a downward shift of the current response at saturation with NMDA, without affecting the EC₅₀. The maximum response obtainable in the absence of peptide could not be achieved by increasing concentrations of NMDA. The same effect was also observed for conantokin inhibition of spermine-potentiated responses. Association rate constants (k(on)) for the peptides were determined in the presence of NMDA and glycine, with and without the addition of spermine. Using a single binding site bimolecular model, k(on) values were 3.1±0.2x10³ M^-1 s^-1 for con-G and 3.2±0.1x10³ M^-1 s^-1 for con-T in the absence of spermine. The added presence of a saturating amount of spermine (300 μM) resulted in an approximate 60% increase in the k(on) values for both con-G and con-T. These results demonstrate that con-T and con-G inhibit NMDA-evoked currents, as well as the potentiation by spermine, in what appears to be a noncompetitive manner, and that spermine increases the rate of conantokin inhibition. Copyright (C) 1999 Elsevier Science Ltd.