Received for publication March 30, 2006.
Revised May 24, 2006.
Accepted for publication June 15, 2006.

Equilibrium constants for NVP-AAM077 acting at recombinant NR1/NR2A and NR1/NR2B NMDA receptors: implications for studies of synaptic transmission

Abstract

We have quantified the effects of the 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 NR1/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 pre-applied or co-applied 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), while 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.

Key words: Glutamate, Ion channel regulation, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Receptor binding studies, Synaptic plasticity

This article has been cited by other articles:

				J. de Marchena, A. C. Roberts, P. G. Middlebrooks, V. Valakh, K. Yashiro, L. R. Wilfley, and B. D. Philpot NMDA Receptor Antagonists Reveal Age-Dependent Differences in the Properties of Visual Cortical Plasticity J Neurophysiol, October 1, 2008; 100(4): 1936 - 1948. [Abstract] [Full Text] [PDF]

				S. L. C. Brothwell, J. L. Barber, D. T. Monaghan, D. E. Jane, A. J. Gibb, and S. Jones NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones J. Physiol., February 1, 2008; 586(3): 739 - 750. [Abstract] [Full Text] [PDF]

				D. C. Wrighton, E. J. Baker, P. E. Chen, and D. J. A. Wyllie Mg2+ and memantine block of rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes J. Physiol., January 1, 2008; 586(1): 211 - 225. [Abstract] [Full Text] [PDF]

				P. E. Chen, M. T. Geballe, E. Katz, K. Erreger, M. R. Livesey, K. K. O'Toole, P. Le, C. J. Lee, J. P. Snyder, S. F. Traynelis, et al. Modulation of glycine potency in rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes J. Physiol., January 1, 2008; 586(1): 227 - 245. [Abstract] [Full Text] [PDF]

				S. Papadia and G. E. Hardingham The Dichotomy of NMDA Receptor Signaling Neuroscientist, December 1, 2007; 13(6): 572 - 579. [Abstract] [PDF]

				F. X. Soriano and G. E. Hardingham Compartmentalized NMDA receptor signalling to survival and death J. Physiol., October 15, 2007; 584(2): 381 - 387. [Abstract] [Full Text] [PDF]

				Q. Chen, S. He, X.-L. Hu, J. Yu, Y. Zhou, J. Zheng, S. Zhang, C. Zhang, W.-H. Duan, and Z.-Q. Xiong Differential Roles of NR2A- and NR2B-Containing NMDA Receptors in Activity-Dependent Brain-Derived Neurotrophic Factor Gene Regulation and Limbic Epileptogenesis J. Neurosci., January 17, 2007; 27(3): 542 - 552. [Abstract] [Full Text] [PDF]