CHARACTERIZATION OF THE SOLUBILIZED NMDA RECEPTOR COMPLEX FROM RAT-BRAIN WITH [H-3] L-689,560, A GLYCINE SITE ANTAGONIST

An antagonist ligand for the glycine site of the NMDA receptor, [H-3]L -689,560, has recently been described. We have investigated the use of this ligand to label NMDA receptors which have been solubilized from rat brain. It has significant advantages over [H-3]dizocilpine ([H-3]M K-801) for this purpose since (a) it is not inhibited by most detergen ts, (b) interactions between the glutamate and glycine sites are maint ained, and (c) equilibrium binding is rapid and of high affinity (K-d = 8.8 +/- 1.9 nm, n = 4). Nevertheless, precautions must be taken to r emove glycine throughout all experimental procedures. In addition we h ave investigated the ability of NMDA receptors to bind to various lect ins and conclude that only N-linked glycosylation is present, consiste nt with consensus sequences for glycosylation present in cloned subuni ts of the NMDA receptor. Further binding of the radioligand [H-3]L-689 ,560 was detected both to the solubilized receptor and to receptor imm obilized on lectin-agarose, identifying it as an appropriate ligand fo r use in the characterization of NMDA receptors during purification pr ocedures.