Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones.
1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight-seal, whole-cell, patch-clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium-sensitive desensitization. With no added glycine, responses to 100 microM-NMDA applied for 1.5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms. 2. Adding glycine to the extracellular solution, over the range 30 nM to 3 microM, both potentiated responses to NMDA and to L-glutamate, and reduced fast desensitization. In the presence of 3 microM-glycine responses to NMDA declined by only 10%. Similar potentiation and reduction of desensitization was obtained with 3 microM concentrations of the glycine analogues D-alanine and D-serine. 3. Analysis of dose-response curves for the action of glycine on responses to 100 microM-NMDA revealed a 3-fold higher potency of glycine for potentiation of peak versus steady-state responses, with concentrations for half-activation of 134 and 382 nM, respectively. The competitive glycine antagonist 7-chlorokynurenic acid produced a similar shift of both the peak and steady-state dose-response curves for glycine, consistent with an equilibrium dissociation constant of 280 nM for interaction of 7-chlorokynurenic acid with the glycine binding site on NMDA receptors. 4. In the presence of 100 nM-glycine, 10 microM-7-chlorokynurenic acid produced nearly complete block of the response to 3 nM-NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex. 5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 microM-glycine. Under these conditions, 7-chlorokynurenic acid produced a concentration-dependent block of both the initial and equilibrium response to NMDA, with a 4-fold greater sensitivity for block of the steady-state current (IC50 = 2.25 microM) than for block of the peak current (IC50 = 8.96 microM). As a result, in the presence of 7-chlorokynurenic acid, responses to NMDA showed strong desensitization, even in the presence of 3 microM-glycine. 6. Our results show that glycine-evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)
Vyklický
L
L
Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, MD 20892.
Benveniste
M
M
Mayer
M L
ML
D000818:Animals; D002478:Cells, Cultured; D004594:Electrophysiology; D005998:Glycine / Q000494:pharmacology*; D006624:Hippocampus / Q000187:drug effects* / Q000502:physiology; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons / Q000187:drug effects*; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects*; D017981:Receptors, Neurotransmitter / Q000187:drug effects
D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D017981:Receptors, Neurotransmitter; D007736:Kynurenic Acid; C057013:7-chlorokynurenic acid; D005998:Glycine