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NEUROPHARMACOLOGY

Identification of Critical Residues in the Amino Terminal Domain of the Human NR2B Subunit Involved in the RO 25-6981 Binding Pocket

Pharma Division, Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland (P.M., V.M., C.B., J.A.K., J.N.C.K.); and Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique Unité Mixte Recherche 8544, Ecole Normale Supérieure, Paris, France (F.P.-D., J.N., P.P.)

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*)--(4-Hydroxyphenyl)--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 (K_D 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.

Address correspondence to: Dr. Pari Malherbe, F. Hoffmann-La Roche Ltd., PRBD-N, Bldg. 69/333, CH-4070 Basel, Switzerland. E-mail: parichehr.malherbe{at}roche.com

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