RECOMBINANT HUMAN NMDA HOMOMERIC NR1 RECEPTORS EXPRESSED IN MAMMALIAN-CELLS FORM A HIGH-AFFINITY GLYCINE ANTAGONIST BINDING-SITE

The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a recept or-channel complex with electrophysiological and pharmacological prope rties characteristic of the N-methyl-D-aspartate receptor. Homomeric N R1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have charac terized, by radioligand binding techniques, the pharmacological proper ties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in hum an embryonic kidney 293 cells. The glycine site antagonist enylaminoca rbonylamino-1,2,3,4-tetrahydroquinoline ([H-3]L-689,560) bound to NR1a - and NR1e-transfected cells with high affinity (K-D = 3.29 and 1.61 n M, respectively). B-max values for NR1a- and NR1e-transfected cells we re 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficien ts were close to unity. K-i values for glycine site antagonists inhibi ting [H-3]L-689,560 binding to NR1e-transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four- to 16-fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist-preferrin g state. K-i values obtained with NR1a-transfected cells were approxim ately twofold lower than those obtained with NR1e-transfected cells. H igh-affinity binding to NR1-transfected cells was not observed with th e transmitter recognition site radioligands L-[H-3]glutamate and lon)- 2-[H-3]amino-4-propyl-5-phosphono-3-pentanoic acid ([H-3]CGP-39653) or the ion-channel radioligand [H-3]dizocilpine ([H-3]MK-801). These res ults indicate that although transfection of mammalian cells with homom eric NR1 recombinant receptors does not appear to result in functional receptors, a glycine binding site is formed that may have a physiolog ical role if present in vivo.