Receptor binding characteristics of the novel NMDA receptor glycine site antagonist [H-3]GV150526A in rat cerebral cortical membranes

Binding of the glycine site antagonist 3-[2-(Phenylamino-carbonyl)ethenyl]- 4,6-dichloro-indole-2-carboxylic acid sodium salt ([H-3]GV150526A) was char acterised in rat cerebral cortical membranes. Saturation experiments indica ted the existence of a high affinity binding site, with a pK(d) value of 9. 08 (K-d = 0.8 nM) and a B-max of 3.4 pmol/mg of protein. A strong linear co rrelation was observed between the displacement potencies for [H-3]GV150526 A and [H-3]glycine of 13 glycine site ligands (r = 0.991). The association kinetics of [H-3]GV150526A binding was monophasic, with a k(on) value of 0. 047 (nM)(-1) min(-1). Dissociation was induced by the addition of an excess of glycine, GV150526A, or 5,7-dichlorokynurenic acid (DCKA), another glyci ne antagonist. With GV150526A and DCKA, the dissociation curves presented s imilar k(off) values (0.068 and 0.069 min(-1), respectively), as expected f rom ligands binding to the same site. Conversely, a significantly lower k(o ff) value (0.027 min(-1)) was found with glycine. Although these data may s uggest that glycine agonists and antagonists bind to discrete sites with an allosteric linkage (rather than interacting competitively), the reason for this difference remains to be elucidated. It is concluded that [H-3]GV1505 26A can be considered a new valuable tool to further investigate the proper ties of the glycine site of the NMDA receptor. (C) 2000 Elsevier Science B. V. All rights reserved.