Substrate exchange properties of the high-affinity glutamate transporter EAAT2.
A stable cell line expressing the predominant brain glutamate transporter EAAT2 was used for the characterization of substrate exchange as a biochemical index for discriminating between substrate and non-substrate inhibitors of the cloned EAAT2 transporter. Addition of 1 mM unlabeled D-aspartate to cells equilibrated with [3H]D-aspartate produced a time-dependent depletion of the [3H] label retained by the cells. L-Aspartate, L-glutamate and L-cysteate produced an equivalent degree of [3H] exchange to that observed with D-aspartate, although the non-substrate EAAT2 inhibitor dihydrokainate and D-glutamate, which does not interact with the substrate binding site, failed to stimulate [3H]D-aspartate exchange. Estimation of EC50 values for the stimulation of [3H] exchange by D-aspartate, L-glutamate and L-trans-2,4-pyrollidine carboxylate (trans-PDC) produced values that were in excellent agreement with the corresponding IC50 values for the same compounds to inhibit EAAT2 uptake. Moreover, trans-PDC was found to produce a lower maximal exchange than that observed with D-aspartate, consistent with the known partial EAAT2 substrate activity of trans-PDC. The estimate of drug induced [3H] efflux with the cloned EAAT2 transporter represents a convenient biochemical assay for the discrimination of substrate and non-substrate inhibitors of the EAAT2 subtype.
Dunlop
J
J
Wyeth Neuroscience, Wyeth-Ayerst Research, Princeton, New Jersey 08543, USA. Dunlopj@war.wyeth.com
D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002490:Central Nervous System / Q000378:metabolism*; D026603:D-Aspartic Acid / Q000378:metabolism / Q000493:pharmacokinetics; D003998:Dicarboxylic Acids / Q000493:pharmacokinetics; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D027342:Excitatory Amino Acid Transporter 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018846:Excitatory Amino Acids / Q000378:metabolism* / Q000493:pharmacokinetics; D018698:Glutamic Acid / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D014179:Neurotransmitter Uptake Inhibitors / Q000493:pharmacokinetics; D011759:Pyrrolidines / Q000493:pharmacokinetics; D011869:Radioligand Assay / Q000379:methods; D013569:Synapses / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D014316:Tritium / Q000493:pharmacokinetics
D003998:Dicarboxylic Acids; D018691:Excitatory Amino Acid Antagonists; D027342:Excitatory Amino Acid Transporter 2; D018846:Excitatory Amino Acids; D014179:Neurotransmitter Uptake Inhibitors; D011759:Pyrrolidines; D014316:Tritium; D018698:Glutamic Acid; D026603:D-Aspartic Acid; C083486:pyrrolidine-2,4-dicarboxylic acid