title: Equilibrium constants for (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) acting at recombinant NR1/NR2A and NR1/NR2B N-methyl-D-aspartate receptors: Implications for studies of synaptic transmission.
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 microM), whereas a value of 2.2 +/- 0.14 microM was obtained with higher (15 microM) 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.
authors.affiliation: Centre for Neuroscience Research, University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Frizelle
authors.forename: Philip E
authors.initials: PE
authors.lastname: Chen
authors.forename: David J A
authors.initials: DJ
authors.lastname: Wyllie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes; D017354:Point Mutation / Q000235:genetics; D011810:Quinoxalines / Q000494:pharmacology*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects*; D014982:Xenopus laevis
chemical_list: C498554:5-(alpha-methyl-4-bromobenzylamino)phosphonomethyl-1,4-dihydroquinoxaline-2,3-dione; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D011810:Quinoxalines; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins