Recombinant Human NMDA Homomeric NR1 Receptors Expressed in Mammalian Cells Form a High‐Affinity Glycine Antagonist Binding Site

Abstract

The cDNA NYMDAR1 (NR1) encodes a single polypeptide that forms a receptor-channel complex with electrophsiological and pharmacological properties characteristic of the N-methyl-D-aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-[3H]phenylaminocarbonylamino - 1,2,3,4-tetrahydroquinoline ([3H]L-689,560) bound to NR1a- and NR1e-transfected cells with high affinity (KD = 3.29 and 1.61 nM, respectively). Bmax values for NR1a- and NR1e-transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. Ki values for glycine site antagonists inhibiting [3H]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-preferring state. Ki values obtained with NR1a-transfected cells were approximately twofold lower than those obtained with NR1e-transfected cells. High-affinity binding to NR1-transfected cells was not observed with the transmitter recognition site radioligands L-[3H]glutamate and D,L-(epsilon)-2-[3H]amino-4-propyl-5-phosphono-3-pentanoic acid ([3H]CGP-39653) or the ion-channel radioligand [3H]dizocilpine ([3H]MK-801).(ABSTRACT TRUNCATED AT 250 WORDS)