OPTIMAL EXPRESSION OF CLONED NMDAR1 NMDAR2A HETEROMERIC GLUTAMATE RECEPTORS - A BIOCHEMICAL-CHARACTERIZATION

The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were express ed transiently either alone or in combination in human embryonic kidne y (HEK) 293 cells. The biochemical and pharmacological properties of t he cloned receptors were compared with those of adult rat brain NMDA r eceptors using both immunological methods with a newly developed anti- NMDAR2A-(1435-1445) antibody and [H-3]MK801 radioligand binding activi ty. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four i mmunoreactive species with M(r)s of 180000, 122000, 97000 and 54000 in rat brain, but only a single band of M(r) 180000 in HEK 293 cells sin gly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cel l membranes yielded a 165000-M, immunoreactive species, which is in ag reement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 2 93 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, inclu ding a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in t he culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [H-3]MK801 binding sites compared with single subunit expression. MK80 1 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR 1 and NMDAR2A receptor subunits co-assemble to form a heteromeric comp lex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity st udies.