Abstract

We have quantified the effects of the N-methyl-d-aspartate (NMDA) receptor antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) at rat recombinant N-methyl-d-aspartate receptor (NR)1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either preapplied or coapplied with glutamate. The IC₅₀ values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses, being 31 ± 2 nM (with glutamate at its EC₅₀ concentration) and 214 ± 10 nM (at 10 times the EC₅₀ concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K_B) of 15 ± 2 nM. Furthermore, Schild analysis of an NMDA receptor carrying a threonine-to-alanine point mutation in the NR2A ligand binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K_B increased by around 15-fold. At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC₅₀ value of 215 ± 13 nM was obtained in the presence of the EC₅₀ concentration of glutamate (1.5 μM), whereas a value of 2.2 ± 0.14 μM was obtained with higher (15 μM) glutamate concentrations. Schild analysis gave a K_B for NVP-AAM077 at NR2B-containing receptors of 78 ± 3 nM. Finally, using a kinetic scheme to model “synaptic-like” activation of NMDA receptors, we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors.