Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322, USA.
J Pharmacol Exp Ther. 2010 Jun;333(3):650-62. doi: 10.1124/jpet.110.166256. Epub 2010 Mar 2.
N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.
N-甲基-D-天冬氨酸（NMDA）受体是配体门控离子通道，介导中枢神经系统兴奋性突触传递中的缓慢、Ca2+通透成分，在突触可塑性、神经元发育和几种神经疾病中发挥关键作用。我们描述了一种基于荧光的测定法，该测定法可测量在稳定表达 NMDA 受体 NR2D 的 BHK-21 细胞系中 NMDA 受体介导的细胞内钙变化，NR1 受四环素诱导启动子（Tet-On）的控制。该测定法通过使用超最大浓度的谷氨酸和甘氨酸来最小化竞争性拮抗剂的检测，从而选择性地识别变构调节剂。该测定法通过成功识别来自两个小型聚焦文库（包括商业上可用的药理学活性化合物文库）的 1800 种筛选化合物中的已知非竞争性但不是竞争性 NMDA 受体拮抗剂而得到验证。通过在非洲爪蟾卵母细胞中表达的重组 NMDA 受体上进行双电极电压钳记录的二次筛选来验证初筛的命中物。该策略确定了几种 NMDA 受体功能的新型调节剂，包括组胺 H3 受体拮抗剂氯苯丙嗪和碘苯丙嗪，以及香草素受体瞬时受体电位阳离子通道，亚家族 V，成员 1（TRPV1）拮抗剂辣椒素。这些化合物是非竞争性拮抗剂，组胺 H3 受体配体对 NR1/NR2B NMDA 受体表现出亚微摩尔效力，这表明可以开发出同时对谷氨酸和组胺受体系统具有高效力的化合物。此外，体内归因于组胺 H3 受体抑制的一些作用也可能涉及 NMDA 受体拮抗作用。
