3H]5,7-Dichlorokynurenic Acid Recognizes two Binding Sites in Rat Cerebral Cortex Membranes

Abstract

Binding of [3H]5,7-dichlorokynurenic acid ([3H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [3H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pKD value of 7.24 (KD = 57.5 nmol/1) and a maximum binding value (Bmax) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [3H]DCKA and [3H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [3H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pKi = 8.24, Ki = 5.6 nmol/1). The low affinity component of [3H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (±)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [3H]DCKA binding site remains to be elucidated.