(±)-threo-3-Methylglutamic acid

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems.[Pubmed:11299317]

J Neurochem. 2001 Apr;77(2):550-7.

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 effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian 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 competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.

Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2.[Pubmed:9614067]

J Biol Chem. 1998 Jun 12;273(24):14698-706.

Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S, 4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.

Contrasting modes of action of methylglutamate derivatives on the excitatory amino acid transporters, EAAT1 and EAAT2.[Pubmed:9145919]

Mol Pharmacol. 1997 May;51(5):809-15.

We have investigated the mechanism of action of a series of glutamate derivatives on the cloned excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), expressed in Xenopus laevis oocytes. The compounds were tested as substrates and competitive blockers of the glutamate transporters. A number of compounds showed contrasting mechanisms of action on EAAT1 compared with EAAT2. In particular, (2S,4R)-4-methylglutamate and 4-methylene-glutamate were transported by EAAT1, with Km values of 54 microM and 391 microM, respectively, but potently blocked glutamate transport by EAAT2, with Kb values of 3.4 microM and 39 microM, respectively. Indeed, (2S,4R)-4-methylglutamate is the most potent blocker of EAAT2 yet described. (+/-)-Threo-3-methylglutamate also potently blocked glutamate transport by EAAT2 (Kb = 18 microM), but was inactive on EAAT1 as either a substrate or a blocker at concentrations up to 300 microM. In contrast to (2S,4R)-4-methylglutamate, L-threo-4-hydroxyglutamate was a substrate for both EAAT1 and EAAT2, with Km values of 61 microM and 48 microM, respectively. It seems that the chemical nature and also the orientation of the group at the 4-position of the carbon backbone of glutamate is crucial in determining the pharmacological activity. The conformations of these molecules have been modeled to understand the structural differences between, firstly, compounds that are blockers versus substrates of EAAT2 and, secondly, the pharmacological differences between EAAT1 and EAAT2.