STABLE EXPRESSION AND CHARACTERIZATION OF RECOMBINANT HUMAN HETEROMERIC N-METHYL-D-ASPARTATE RECEPTOR SUBTYPES NMDAR1A 2A AND NMDAR1A/2B INMAMMALIAN-CELLS/

The electrophysiological and pharmacological properties of two mammali an cell lines stably transfected with cDNAs encoding recombinant human N-methyl-D-aspartate (NMDA) receptor subtypes NMDAR1A/2A and NMDAR1A/ 2B are described. In whole-cell electrophysiological recordings, appli cation of NMDA/glycine elicited inward currents at negative holding po tentials in human NMDAR1A/2A (hNMDAR1A/ 2A)- and hNMDAR1A/2B-expressin g cells. The current-voltage relationships determined in both cell lin es in the presence and absence of external Mg++ were similar to those observed with recombinant rat NMDA receptors. Power spectra calculated from NMDA/glycine-induced currents for both NMDA receptor-expressing cell lines suggested a kinetically homogeneous population of channels. Immunoprecipitation with an anti-NMDAR1A antibody coprecipitated the corresponding NMDAR2 subunit with the NMDAR1A, suggesting that heterom eric complexes are formed in these stable cell lines. Stimuration of N MDA receptors evoked an increase in intracellular Ca++, which was used to characterize their pharmacological properties. NMDA displayed less intrinsic activity than did glutamate in both NMDA receptor-expressin g cell lines and was a 4-fold more potent agonist at hNMDAR1A/2B than hNMDAR1A/ 2A. NMDA/glycine-evoked increases in Ca++ levels were in hib ited by CGS 19755, )-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, MK-801, ketamine and ifenprodil. -)-3-(2-Carboxypiperazin-4-yl)propyl- 1-phosphonate was a 3-fold more potent antagonist at hNMDAR1A/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNM DAR1A/2B, being 250-fold more potent than against hNMDAR1A/2A. These d ata suggest that cells stably expressing recombinant heteromeric hNMDA R1A/2A and hNMDAR1A/ 2B represent pharmacologically valid experimental systems to study human NMDA receptors.