Kinetic Characterization of the Phencyclidine-N-Methyl-D-aspartate Receptor Interaction: Evidence for a Steric Blockade of the Channel

The nature of the interactions between the N-methyl-D-aspartate (NMDA) and the phencyclidine (PCP) receptors was studied in membranes obtained from rat cerebral cortex and washed repeatedly to remove endogenous excitatory amino acids. Binding of [³H]-N-[1 -(2-thienyl)cyclohexyl]piperidine ([³H]TCP) to its receptor sites in these membranes proceeded slowly and did not reach equilibrium even after incubation for 4 h at 25 °C. The dissociation rate of [³H]TCP-receptor complexes was also slow (t_1/2= 128–165 min). Both association and dissociation followed first-order reaction kinetics, with similar time constants (0.0054 min^-1). Addition of glutamate and glycine to the washed membranes was immediately followed by a marked increase in the rates of both association of [³H]TCP with the receptors and its dissociation from them (t_1/2= 8 min). Association now followed second-order reaction kinetics. Accelerated association of [³H]TCP with its binding sites could also be induced by NMDA or by glutamate alone, and glycine enhanced the effect. All effects of glutamate and glycine on [³H]TCP binding kinetics were blocked by the competitive NMDA receptor antagonist AP-5 [D-(-)-2-amino-5-phosphovaleric acid], [³H]TCP-receptor interactions at equilibrium were not altered by AP-5 or by glutamate and glycine. The binding data were fitted to a model in which interactions of [³H]TCP with the receptor involve a two-step process: the outside ligand must cross a barrier (presumably a closed NMDA receptor channel in the absence of agonists). Once agonists are added, this limitation is removed (presumably because the channel is open). The excellent agreement between the kinetic and equilibrium binding parameters with the predictions of our model, as well as with previous electrophysiological data on the mode of noncompetitive blocking of the NMDA receptor channel by PCP-like drugs, suggests that these drugs are steric blockers of the channel and prefer its open state.