Abstract

dl-threo-β-Benzyloxyaspartate (dl-TBOA), a novel derivative ofdl-threo-β-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters.dl-TBOA inhibited the uptake of [¹⁴C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (K _i = 42 μm) with almost the same potency asdl-threo-β-hydroxyaspartate (K _i = 58 μm). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of dl-TBOA (K _i = 5.7 μm) was much more potent than that of dihydrokainate (K _i = 79 μm), which is well known as a selective blocker of this subtype. Electrophysiologically, dl-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by addingdl-TBOA without a significant change in the maximum current. The K _b values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 μm and 116 nm, respectively. These results demonstrated that dl-TBOA is, so far, the most potent competitive blocker of glutamate transporters.dl-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover,dl-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, dl-TBOA should be a useful tool for investigating the physiological roles of transporters.