Conantokin-G (con-G) and conantokin-T (con-T) are small (17 and 21 amino acids, respectively) gamma-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 microM) in a dose-dependent manner while maintaining a holding potential of -70 mV. In the presence of saturating amounts of NMDA (100 microM) and glycine (1 microM), the IC50 values obtained were 487 +/- 85 nM for con-G and 1030 +/- 130 nM for con-T. NMDA (10 microM-1 mM) dose-response curves produced in the presence of con-G or con-T (1 or 3 microM) 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 +/- 0.2 x 10(3) M(-1) s(-1) for con-G and 3.2 +/- 0.1 x 10(3) M(-1) s(-1) for con-T in the absence of spermine. The added presence of a saturating amount of spermine (300 microM) 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.