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

Abstract Binding of the glycine site antagonist 3-[2-(Phenylamino-carbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt ([ 3 H]GV150526A) was characterised in rat cerebral cortical membranes. Saturation experiments indicated 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 correlation was observed between the displacement potencies for [ 3 H]GV150526A and [ 3 H]glycine of 13 glycine site ligands ( r =0.991). The association kinetics of [ 3 H]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 glycine antagonist. With GV150526A and DCKA, the dissociation curves presented similar k off values (0.068 and 0.069 min −1 , respectively), as expected from ligands binding to the same site. Conversely, a significantly lower k off value (0.027 min −1 ) was found with glycine. Although these data may suggest 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 [ 3 H]GV150526A can be considered a new valuable tool to further investigate the properties of the glycine site of the NMDA receptor.