GENERATION AND CHARACTERIZATION OF STABLE CELL-LINES EXPRESSING RECOMBINANT HUMAN N-METHYL-D-ASPARTATE RECEPTOR SUBTYPES

Transfection of mouse L(tk-) cells with human N-methyl-D-aspartate (NM DA) receptor subunit cDNAs under the control of a dexamethasone-induci ble promoter has been used to generate two stable cell lines expressin g NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B rece ptors, The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors deter mined by radioligand binding techniques. Pharmacological differences w ere identified between the two NMDA receptor subtypes. The glutamate s ite antagonist lon)-2-[H-3]amino-4-propyl-5-phosphono-3-pentanoic acid ([H-3]CGP 39653) had high affinity for NR1a/NR2A receptors (K-D = 3.9 3 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists s howed a 2.6-5.4-fold higher affinity for NR1a/NR2B receptors. Data fro m radioligand binding studies indicated that one of the cell lines, NR 1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, whic h may exist as homomeric assemblies, This observation has implications when interpreting data from pharmacological analysis of recombinant r eceptors, as well as understanding the assembly and control of express ion of native NMDA receptors.