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Characterization of the Tritium-Labeled Analog of L-threo--Benzyloxyaspartate Binding to Glutamate Transporters

Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, Japan (K.S., Y.Y.-K.); Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan (Y.O., S.K., T.N.); Yasuda Women's University, Hiroshima, Japan (M.S.); and National Institute of Advanced Industrial Science and Technology, Midorigaoka, Ikeda, Japan (Y.S.)

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Termination of glutamate receptor activation and maintenance of low extracellular glutamate concentrations are primarily achieved by glutamate transporters (excitatory amino acid transporters 1-5, EAATs1-5) located on both the nerve endings and the surrounding glial cells. To identify the physiological roles of each subtype, subtype-selective EAAT ligands are required. In this study, we developed a binding assay system to characterize EAAT ligands for all EAAT subtypes. We recently synthesized novel analogs of threo--benzyloxyaspartate (TBOA) and reported that they blocked glutamate uptake by EAATs 1-5 much more potently than TBOA. The strong inhibitory activity of the TBOA analogs suggested that they would be suitable to use as radioisotope-labeled ligands, and we therefore synthesized a tritiated derivative of (2S,3S)-3-{3-[4-ethylbenzoylamino]benzyloxy}aspartate ([³H]ETB-TBOA). [³H]ETB-TBOA showed significant high-affinity specific binding to EAAT-transfected COS-1 cell membranes with each EAAT subtype. The Hill coefficient for the Na⁺-dependence of [³H]ETB-TBOA binding revealed a single class of noncooperative binding sites for Na⁺, suggesting that Na⁺ binding in the ligand binding step is different from Na⁺ binding in the substrate uptake process. The binding was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with glutamate uptake assay results reported previously. Thus, the [³H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.

Address correspondence to: Dr. Keiko Shimamoto, Suntory Institute for Bioorganic Research, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503. E-mail: shimamot{at}sunbor.or.jp

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