Characterization of novel L-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters
Characterization of novel L-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters
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DOI：
10.1124/mol.65.4.1008
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发表时间：
2004-04-01
影响因子：
3.6
通讯作者：
Shigeri, Y
中科院分区：
文献类型：
--
作者：
Shimamoto, K;Sakai, R;Shigeri, Y
谷氨酸转运体的不可转运阻滞剂是研究突触传递机制的重要工具。dl -三- β -苯氧天冬氨酸（DL-TBOA）是所有兴奋性氨基酸转运体（EAATs）亚型的有效阻滞剂。我们表征了新的L-TBOA类似物在它们各自的苯环上具有取代基。这些类似物显著抑制了标记谷氨酸的摄取，其中(2S,3S)-3-{3-[4-（三氟甲基）苯甲酰氨基]苯氧基}天冬氨酸（TFB-TBOA）的抑制作用最强。在瞬时表达EAATs的细胞摄取实验中，tbb - tboa对EAAT1、EAAT2和EAAT3的IC50值分别为22、17和300 nM。与L-TBOA相比，tbb - tboa对EAAT1和EAAT2的抑制作用明显更强（EAAT1-3的IC50值分别为33、6.2和15 muM）。电生理分析显示，TBOA类似物阻断了所有5种EAAT亚型的运输相关电流，也阻断了EAAT5的泄漏电流。抑制底物诱导电流的类似物的等级顺序与摄取试验中观察到的相同。然而，TFB-TBOA的动力学与L-TBOA的动力学不同，这可能是由于TFB-TBOA具有较强的结合亲和力。值得注意的是，TFB-TBOA不影响其他具有代表性的神经递质转运体或受体，包括嗜离子性和代谢性谷氨酸受体，这表明它对EAATs具有高度选择性。此外，脑室内给予TBOA类似物可引起小鼠严重的惊厥行为，可能是由于谷氨酸的积累。综上所述，这些发现表明新的TBOA类似物，特别是TFB-TBOA，应该作为阐明谷氨酸转运体生理作用的有用工具。
Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-beta-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their respective benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC50 values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC50 values for EAAT1-3 were 33, 6.2, and 15 muM, respectively). Electrophysiological analyses revealed that TBOA analogs block the transport-associated currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that observed in the uptake assay. However, the kinetics of TFB-TBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters.