[H-3]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

Activation of NMDA receptors requires the presence of glycine as a coagonis t which binds to a site that is allosterically linked to the glutamate bind ing site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [H-3]CGP 61594 was synthesized. In reversib le binding assays using crude rat brain membranes, [H-3]CGP 61594 labeled w ith high affinity (K-D = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of vario us glycine site ligands and the allosteric modulation of [H-3]CGP 61594 bin ding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a gl ycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specif ically photolabeled by [H-3]CGP 61594 in brain membrane preparations. The p hotolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [H-3]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. I t will serve as a tool for the identification of structural elements that a re involved in the formation of the glycine binding domain of NMDA receptor s in situ and will thereby complement the mutational analysis of recombinan t receptors. (C) 1999 Elsevier Science Ltd. All rights reserved.