Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible...

Pharmacological characterization of N-methyl-D-aspartate (NMDA) receptors has been hampered by the difficulty to outwit cytotoxicity after functional expression in recombinant systems. In this study a muristerone-inducible expression system for the NNMDA-R1 subunit was used. This was combined with constitutive expression of NMDA-R2A, 2B, 2C and 2D in different cell clones. After establishment of the cell lines, quantitative RT-PCR demonstrated the inducibility of the NNMDA-R1 subunit, and verified the expression of the NMDA-R2 subunits in the different cell clones. Functional responses were characterized using calcium influx through the ion channel as a robust assay system. Stimulation of the NMDA-receptor subtypes in the different cell lines led to calcium transients which were rising gradually, peaked after 30-160 s and declined thereafter very slowly. The expression of the four different NMDA-receptor subtypes in the same cellular background allowed a direct pharmacological comparison of the different receptors. Glutamate showed the highest potency at the NMDA-R1-2D. NMDA displayed at all subtypes a lower potency compared to glutamate and was a partial agonist except at the NMDA-R1-2D. 20 antagonists were tested in this study and the pharmacological characterization of the inhibition of glutamate-evoked elevation of intracellular free Ca(2+) revealed a distinct rank order of antagonist potency for each receptor subtype. These data illustrate that assessment of calcium transients upon receptor stimulation in the same cellular background is a powerful tool to compare the functional effects of compounds acting at the different NMDA-R2 receptors.