Malherbe Pari, Mutel Vincent, Broger Clemens, Perin-Dureau Florent, Kemp John A, Neyton Jacques, Paoletti Pierre, Kew James N C
Pharma Division, Discovery Research CNS, F-Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
J Pharmacol Exp Ther. 2003 Dec;307(3):897-905. doi: 10.1124/jpet.103.056291. Epub 2003 Oct 8.
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.
N-甲基-D-天冬氨酸（NMDA）受体在生理过程中发挥关键作用，尤其是在突触可塑性方面，同时也在诸如癫痫和急性神经退行性变等神经病理状态中起作用。R-（R*，S*）-α-（4-羟基苯基）-β-甲基-4-（苯基甲基）-1-哌啶丙醇（RO 25-6981）是一种对含有NR2B亚基的NMDA受体具有高亲和力和选择性的阻滞剂。通过定点诱变、[3H]RO 25-6981结合实验、非洲爪蟾卵母细胞电压钳记录以及分子建模，我们在人NR2B亚基N端类LIVBP结构域内确定了几个参与RO 25-6981结合位点的关键残基。两个突变体NR2B（D101A）和NR2B（F176A）导致[3H]RO 25-6981结合完全丧失，并且也消除了RO 25-6981介导的对NMDA诱导电流的高亲和力抑制作用。突变体NR2B（T233A）导致结合亲和力显著降低13倍。F182A、D104A或K234A突变对结合亲和力的影响较为适中（KD值分别增加8倍、7倍和6倍）。基于mGlu1受体氨基端结构域的X射线晶体结构构建的NR2B类LIVBP结构域三维模型中，关键残基位于中央裂隙处，与RO 25-6981的相互作用可能稳定该结构域的闭合结构。我们的结果表明，天冬氨酸-101、苯丙氨酸-176和苏氨酸-233这三个氨基酸是RO 25-6981与人NR2B类LIVBP结构域高亲和力结合的重要分子决定因素。文中还提出了RO 25-6981可能的结合模式。
