Received for publication May 30, 2006.
Revised October 15, 2006.
Accepted for publication October 17, 2006.

Characterization of the Tritium Labeled Analog of L-Threo-beta-benzyloxyaspartate (TBOA) Binding to Glutamate Transporters

Abstract

L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). 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 both on the nerve endings and the surrounding glial cells. In order 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 EAATs1-5 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⁺-dependency of [³H]ETB-TBOA binding revealed a single class of non-cooperative 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 of [³H]ETB-TBOA was displaced by known substrates and blockers. The rank order of inhibition by these compounds was consistent with the previously reported glutamate uptake assay results. Thus, the [³H]ETB-TBOA binding assay will be useful to screen novel EAAT ligands for all EAAT subtypes.

Key words: Amino Acid, Receptor binding studies, Excitotoxicity, neurodegeneration

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