title: Identification of critical residues in the amino terminal domain of the human NR2B subunit involved in the RO 25-6981 binding pocket.
abstract: N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-alpha-(4-Hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.
authors.affiliation: Pharma Division, Discovery Research CNS, F-Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. parichehr.malherbe@roche.com
authors.forename: Pari
authors.initials: P
authors.lastname: Malherbe
authors.forename: Vincent
authors.initials: V
authors.lastname: Mutel
authors.forename: Clemens
authors.initials: C
authors.lastname: Broger
authors.forename: Florent
authors.initials: F
authors.lastname: Perin-Dureau
authors.forename: John A
authors.initials: JA
authors.lastname: Kemp
authors.forename: Jacques
authors.initials: J
authors.lastname: Neyton
authors.forename: Pierre
authors.initials: P
authors.lastname: Paoletti
authors.forename: James N C
authors.initials: JN
authors.lastname: Kew
mesh_terms: D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010636:Phenols / Q000378:metabolism*; D010880:Piperidines / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; D018691:Excitatory Amino Acid Antagonists; C121001:NR2B NMDA receptor; D010636:Phenols; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; C109643:Ro 25-6981