title: The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures.
abstract: Cysteine availability is normally the rate-limiting factor in glutathione synthesis. How neurons obtain cysteine from extracellular space is not well established. Here we used mouse cortical neuron cultures to examine the role of the excitatory amino acid transporters (EAATs) in neuronal cysteine uptake. The cultured neurons expressed both EAAT2 and EAAT3. Cysteine uptake was predominantly (> 85%) Na+-dependent, with an apparent Km of 37 microm. Cysteine uptake was reduced by the EAAT substrates l-glutamate and l-aspartate and by synthetic EAAT inhibitors. The non-selective EAAT inhibitor threo-beta-hydroxyaspartate had a significantly greater maximal inhibitory effect than did the EAAT2-selective inhibitor, dihydrokainate, indicating uptake by both EAAT2 and EAAT3. Serine, a substrate of ASC uptake system, had negligible effects on cysteine uptake at 10-fold excess concentrations. To assess the functional importance of EAAT-mediated cysteine uptake in neuronal glutathione synthesis, cultures were treated with diethylmaleate to deplete glutathione, then incubated with cysteine in the presence or absence of EAAT inhibitors. Threo-beta-benzyloxyaspartate and the non-transportable inhibitor threo-beta-hydroxyaspartate both inhibited the cysteine-dependent glutathione synthesis. The findings suggest that neuronal EAAT activity can be a rate-limiting step for neuronal glutathione synthesis and that the primary function of EAATs expressed by neurons in vivo may be to transport cysteine.
authors.affiliation: Department of Neurology, University of California at San Francisco and Veterans Affairs Medical Center, San Francisco, California 94121, USA.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Chen
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Swanson
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001253:Astrocytes / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D003545:Cysteine / Q000378:metabolism / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D027341:Excitatory Amino Acid Transporter 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D027342:Excitatory Amino Acid Transporter 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000494:pharmacology; D005978:Glutathione / Q000096:biosynthesis; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D027322:Amino Acid Transport System X-AG; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; C493399:Slc1a1 protein, mouse; C493396:Slc1a2 protein, mouse; D027981:Symporters; C120673:benzyloxyaspartate; C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; C020889:dihydrokainic acid; D012964:Sodium; D005978:Glutathione; D003545:Cysteine; D007608:Kainic Acid