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Abstract

Glycine is an essential co-agonist of the excitatory N-methyl-d-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor family. The glycine binding site of this hetero-oligomeric ion channel protein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit mediate glutamate binding. Here, segments S1 and S2 of the NR1 polypeptide were fused via a linker peptide followed by N- and C-terminally tagging with Flag and His₆ epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine binding site construct resulted in efficient secretion of a soluble fusion protein of about 53 kDa. After affinity purification to near-homogeneity, the fusion protein bound the competitive glycine site antagonist [³H]MDL105,519 with high affinity (K_d = 5.22 ± 0.13 nm) similar to that determined with rat brain membrane fractions. This high affinity binding could be competed by the glycine site antagonist 7-chlorokynurenic acid as well as the agonists glycine and d-serine but not by l-glutamate. This indicates that the S1 and S2 domains of the NR1 subunit are sufficient for the formation of a glycine binding site that displays pharmacological properties similar to those of the NMDA receptorin vivo.