Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na+- dependent excitatory amino acid transporter (EAAT). We have examined the ef fects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expresse d in Xenopus oocytes and on the transport of radiolabeled substrate in mamm alian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitivel y inhibited uptake of D-[H-3]-aspartate into both cortical and cerebellar s ynaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substra te-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG w as unable to elicit heteroexchange of preloaded D-[H-3]-aspartate in cerebe llar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cult ured hippocampal neurons: concentrations greater than 100 muM T3MG were req uired to elicit significant NMDA receptor-mediated currents. Thus, T3MG rep resents a pharmacological tool for the study of not only the predominant EA AT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.