| Research Abstract |
Non-transportable blockers for the glutamate transporters are indispensable tools for investigating mechanisms of synaptic transmission. DL-threo-β-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 its benzene ring. The analogs significantly inhibited labeled glutamate uptake with (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA) being the most potent among them. In the uptake assay using cells transiently expressing EAATs, the IC_<50> values ties of TFB-TBOA for EAATI, EAAT2, and EAAT3 were 22 nM, 17 nM, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAATI and EAAT2 compared with L-TBOA (IC_<50> values for EAATs1-3 were 33 μM, 0.2μM, and 15 μM, 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 potency for inhibiting substrate-induced currents was identical to that observed with the uptake assay. However, the kinetic character of TFB-TBOA was different from that 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, which would be attributed to the accumulation of glutamate. Taken together, new TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters.
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