The glycine site modulates NMDA-mediated changes of intracellular free calcium in cultures of hippocampal neurons

Abstract Recent evidence indicates that the N- methyl- d -aspartate (NMDA) receptor-channel complex contains a glycine subunit whose activation may be necessary for channel operation. It has been previously shown that stimulation of the NMDA receptor leads to an increase in intracellular ionic Ca 2+ ([Ca 2+ ] i ); therefore, we examined the role of the NMDA receptor-associated glycine site in modulating [Ca 2+ ] i using the fluorescent dye Fura II in hippocampal neuron cultures. A 3-s pulse of 200 μM NMDA resulted in a mean [Ca 2+ ] i increase of 363 nM above the average resting concentration of 122 nM. Perfusion of the glycine site antagonist 7-chlorokynurenate (Cl-Kyn) essentially eliminated the NMDA-induced alteration in [Ca 2+ ] i . Either 40 μM glycine or 50 μM d -serine completely reversed the effect of Cl-Kyn, indicating that the drug was acting at the glycine site. The NMDA receptor antagonists 2-amino-5-phosphonovalerate (AP5) and ketamine, which bind to the glutamate recognition site and the ion channel, respectively, also blocked the NMDA-mediated [Ca 2+ ] i response; however, glycine or d -serine did not reverse this effect. These data show that the glycine binding site coupled to the NMDA receptor modulates the NMDA-mediated increase in [Ca 2+ ] i . Antagonists of the glycine site provide a new tool to investigate and possibly control neuroplasticity and neurotoxicity related to the NMDA receptor complex.