Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterization.
The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex 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 studies.
Cik
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Department of Pharmaceutical Chemistry, School of Pharmacy, London, U.K.
Chazot
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PL
Stephenson
F A
FA
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D000595:Amino Acid Sequence; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011810:Quinoxalines; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione