1. Voltage-clamp experiments were used to study N-methyl-d-aspartic acid (NMDA) receptor antagonists applied by fast perfusion to mouse hippocampal neurons in dissociated culture. 2. Preincubation with the NMDA antagonists zinc (3-30 μM) and magnesium (30-300 μM) reduced subsequent responses to 100 μM NMDA applied together with these antagonists. No time dependence of antagonism was observed when responses were measured at the start and at the end of NMDA pulses 1.25-1.5 s in duration. 3. Two competitive antagonists of simular affinity in equilibrium experiments, D-2-amino-5-phosphonopentanoic acid (D-AP5) and 3-((±)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), had different profiles of action when applied as described above. With d-AP5, pulses of NMDA produced fast-on, fast-off responses, of reduced amplitude, simular to the effect of Zn and Mg. Responses to NMDA in the presence of CPP were also of reduced amplitude but, in addition, showed slow activation, such that the antagonist action of CPP decreased with time after the application of NMDA. 4. In the presence of 3 μM glycine and NMDA receptor antagonists with activity at the glycine modulatory site, either kynurenic acid (Kyn), 7-chlorokynurenic acid (7Cl-Kyn), or 5-chloro-indole-2-carboxylic acid (5Cl-12CA), NMDA-evoked responses showed apparent use-dependent antagonism, such that the peak response to NMDA was much greater than the equilibrium response. A similar effect was produced by preincubation with low concentrations of glycine (<300 nM), which enhances desensitization of responses to NMDA. The apparent use-dependent action of glycine antagonists could be reversed on raising the glycine concentration and did not vary appreciably with changes in membrane potential over the range -60 to +50 mV. 5. Concentration-jump application of NMDA antagonists, in the presence of 100 μM NMDA and 3 μM glycine, were used to study antagonist association and dissociation kinetics directly. For D-AP5 and CPP, the dissociation rate was independent of antagonist concentration, and ~15 times faster for D-AP5 (19.6 s-1) than for CPP (1.36 s-1). The association rate for D-AP5 and CPP increased with antagonist concentration in a linear manner over the range 3-30 μM and was slower for CPP than for D-AP5, consistent with their similar potency at equilibrium. 6. In contrast to results obtained with CPP and D-AP5, the association rate for 7Cl-Kyn was ~3 times slower than the dissociation rate and did not change with concentration of antagonist. We suggest that this is because dissociation of glycine from NMDA receptors must occur prior to binding of 7Cl-Kyn and that slow dissociation of glycine delays the onset of action of 7Cl-Kyn. Consistent with this, in the presence of L-analine - a glycine agonist that has a fast dissociation rate - the kinetics of antagonism of 7 Cl-Kyn were conventional: the association rate for 7 Cl-Kyn was faster than the dissociation rate and increased with antagonist concentration in a linear manner over the range 3-30 μM 7 Cl-Kyn. 7. Our results with 7Cl-Kyn and 5Cl-12CA confirm the role of glycine in regulating NMDA receptor desensitization and show that attempts to measure the association rate for glycine antogonists are limited by the slow dissociation of glycine from the NMDA receptor, unless lower affinity analogues such as L-alanine are used in place of glycine. 8. Kinetic experiments with competitive NMDA antagonists reveal features not apparent from equilibrium studies. For D-AP5 and CPP, the slow dissociation rate of CPP must be balanced by a reduced association rate, such that at equilibrium CPP is only slightly more potent than D-AP5. Were this not the case, CPP would be a considerably more potent NMDA antagonist. It is possible that during binding of CPP, conformational restrictions imposed by the piperazine ring limit the speed at which CPP can bind to the NMDA receptor. In contrast, D-AP5 is a relatively unconstrained molecule, and this might contribute to faster binding kinetics.
