Identification of critical residues in the amino terminal domain of the human NR2B subunit involved in the RO 25-6981 binding pocket.
Résumé
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.