Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

A 45 Ca 2+ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N-methyl-D-aspartate (NMDA) receptors. Inhibition of glutamate-stimulated 45 Ca 2+ influx by six glycine-site antagonists and inhibition of glycine-stimulated 45 Ca 2+ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated 45 Ca 2+ influx for recombinant NMDA receptors, inhibiting and stimulating 45 Ca 2+ influx into cells expressing NR1a/NR2A receptors (IC 50 = 408 μM) and NR1a/NR2B receptors (EC 50 = 37.3 μM), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC 50 = 0.099 μM) compared with NR1a/NR2A receptors (IC 50 = 164 μM). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg 2+ and Zn 2+ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of 45 Ca 2+ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.