Characterization of the solubilized NMDA receptor complex from rat brain with [³H]L-689,560, a glycine site antagonist

Abstract

An antagonist ligand for the glycine site of the NMDA receptor, [³H]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 [³H]dizocilpine ([³H]MK-801) for this purpose since (a) it is not inhibited by most detergents, (b) interactions between the glutamate and glycine sites are maintained, 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 remove glycine throughout all experimental procedures. In addition we have investigated the ability of NMDA receptors to bind to various lectins and conclude that only N-linked glycosylation is present, consistent with consensus sequences for glycosylation present in cloned subunits of the NMDA receptor. Further binding of the radioligand [³H]L-689,560 was detected both to the solubilized receptor and to receptor immobilized on lectin-agarose, identifying it as an appropriate ligand for use in the characterization of NMDA receptors during purification procedures.

Key Words:

• NMDA receptor
• [³H]L-689,560
• glycine site
• solubilization