Evaluation and biological properties of reactive ligands for the mapping of the glycine site on the N-methyl-D-aspartate (NMDA) receptor

The glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, given its potential as pharmacological target, has been thoroughly studied by st ructure-activity relationships, which has made possible its description in terms of spatial limits and interactions of various types. A structural mod el, based on mutational analysis and sequence alignements, has been propose d. Yet, the amino acid residues responsible for the interactions with the l igand have not been unambiguously characterized. To evidence nucleophilic p ocket-lining residues, we have designed and synthesized reactive glycine-si te ligands derived from 3-substituted 4-hydroxy-quinolin-2(1H)-ones by intr oducing various electrophilic groups at different positions of the molecule . These ligands were found to have high affinity at the glycine site and to be functional antagonists by inhibiting glycine/glutamate-induced currents in transfected oocytes. The correlation between their potency and their su bstitution pattern was strictly consistent with previously established stru cture-activity relationships. Most ligands displayed intrinsic reactivity t oward cysteine, but none inactivated wild-type receptors. This is consisten t with the model since it indicates the absence of exposed cysteine in the glycine-binding site. A strategy of cysteine incorporation by point mutatio ns at selected polypeptide positions will create unambiguously localized ta rgets for our reactive probes.