1Department of Neuropharmacology, Institute of Nautical Medicine, Nantong University, Nantong 226001, China
2Department of Neurology, Affiliated Hospital, Nantong University, Nantong 226007, China

Abstract 

Objective Glycine acts as a co-agonist for the activation of N-methyl-D-aspartate receptors (NMDARs) by binding to glycine sites, thus potentiating glutamate-elicited responses and inhibiting NMDAR desensitization in a dosedependent manner. The present study aimed to characterize the glycine-dependent inactivation of NMDARs and to explore its pathophysiological significance. Methods Primary hippocampal cell cultures from embryonic days 17–18 rats were treated with NMDA or NMDA plus glycine. Patch-clamp recording and intracellular Ca2+ imaging were performed to test the effects of glycine on NMDA-activated currents and increase of intracellular free Ca2+ respectively. Immunofluorescence staining was conducted to examine NR1 internalization. Cell damage was tested with MTT method and lactate ehydrogenase leakage. Results Glycine reduced the peak current and Ca2+ influx elicited by NMDA application at concentrations ≥300 μmol/L. This is a novel suppressive influence of glycine on NMDAR function, since it occurs via the NMDAR glycine-binding site, in contrast to the classic suppression, which occurs through the binding of glycine to glycine receptors. The level of membrane NMDARs was measured to evaluate whether internalization was involved. Immunohistochemical labeling showed that incubation with high concentrations of NMDA plus glycine did not change the expression of NMDARs on the cell surface
when compared to the expression without glycine; hence the possibility of NMDAR internalization primed by glycine binding was excluded. Conclusion In summary, the novel suppressive effect of glycine on NMDARs was mediated via binding to the glycine site of the NMDAR and not by activation of the strychnine-sensitive glycine-receptor-gated chloride channel or by the internalization of NMDARs. The inhibitory influence of glycine on NMDARs adds a new insight to our knowledge of the complexity of synaptic transmission.

Keywords

glycine; N-methyl-D-aspartate receptors; calcium imaging; NMDA-NR1 subunit; hippocampal neurons; inactivation

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