[H-3]5,7-DICHLOROKYNURENIC ACID RECOGNIZES 2 BINDING-SITES IN RAT CEREBRAL-CORTEX MEMBRANES

Binding of [H-3]5,7-dichlorokynurenic acid ([H-3]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl- D-aspartate (NMDA) receptor channel complex, was characterized in syna ptic plasma membranes from rat cerebral cortex. Non linear curve fitti ng of [H-3]DCKA saturation and homologous displacement isotherms indic ated the existence of two binding sites: a specific, saturable, high a ffinity site, with a pK(D) value of 7.24 (K-D = 57.5 nmol/l) and a max imum binding value (B-max) of 6.9 pmol/mg of protein and a second site , with micromolar affinity. The pharmacological profile of both bindin g components was determined by studying the effect on [H-3]DCKA and [H -3]glycine binding of a series of compounds known to interact with dif ferent excitatory and inhibitory amino acid receptors. These studies c onfirmed the identity of the high affinity site of [H-3]DCKA binding w ith the strychnine-insensitive glycine site of the NMDA receptor chann el complex. carbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid so dium salt (GV 150526A), a new, high affinity, selective glycine site a ntagonist (1), was the most potent inhibitor of this component of bind ing (pK(i) = 8.24, K-i = 5.6 nmol/l). The low affinity component of [H -3]DCKA binding was insensitive to the agonists glycine and D-serine a nd 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 [H-3]DCKA binding site remains to be elucida ted.