Reactive affinity probes for the mapping of the glycine-binding site of the NMDA receptor NR1 subunit

The glycine co-agonist binding site of the NMDA receptor is a target for th e prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, h ave been investigated by ligand structure-activity relationships and by sit e-directed mutagenesis. On the basis of a structural model which is current ly proposed for this site, we have designed and synthesized six affinity ma rkers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with H-3-DCKA binding to rat brain membranes at equilibri um with nanomolar to low-micromolar affinities, and antagonize glycine-evok ed currents in oocytes transfected with wild-type NR1-NR2B. However, they d o not induce a time-shift in binding equilibria, and do not inactivate irre versibly the glycine evoked currents. Since they react only with cysteine a t physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent poten tial topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.