title: Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism.
abstract: Glutamate uptake into nerve cells and astrocytes via high-affinity transporters controls the extracellular glutamate concentration in the brain, with major implications for physiological excitatory neurotransmission and the prevention of excitotoxicity. We report here that three recently cloned rat glutamate transporter subtypes, viz. EAAC1 (neuronal), GLT1 and GLAST (glial), possess a redox-sensing property, undergoing opposite functional changes in response to oxidation or reduction of reactive sulphydryls present in their structure. In particular, thiol oxidation with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) and disulphide reduction with dithiothreitol (DTT) result, respectively, in reduced and increased uptake capacity by a preparation of partially purified brain transporters as well as by the three recombinant proteins reconstituted into liposomes. In this model system, EAAC1, GLT1 and GLAST react similarly to DTT/DTNB exposures despite their different contents of cysteines, suggesting that only the conserved residues might be involved in redox modulation. Redox sensitivity is a property of the glutamate transporters also when present in their native cell environment. Thus, by using cultured cortical astrocytes and the whole-cell patch-clamp technique we were able to observe dynamic increase and decrease of the glutamate uptake current in response to application of DTT and DTNB in sequence. Moreover, in the same paradigm, DDT-reversible current inhibition was observed with hydrogen peroxide instead of DTNB, indicating that the SH-based redox modulatory site is targeted by endogenous oxidants and might constitute an important physiological or pathophysiological regulatory mechanism of glutamate uptake in vivo.
authors.affiliation: Institute of Pharmacological Sciences, Centre of Neuropharmacology, University of Milan, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Trotti
authors.forename: B L
authors.initials: BL
authors.lastname: Rizzini
authors.forename: D
authors.initials: D
authors.lastname: Rossi
authors.forename: O
authors.initials: O
authors.lastname: Haugeto
authors.forename: G
authors.initials: G
authors.lastname: Racagni
authors.forename: N C
authors.initials: NC
authors.lastname: Danbolt
authors.forename: A
authors.initials: A
authors.lastname: Volterra
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001921:Brain / Q000166:cytology; D001923:Brain Chemistry / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis; D004594:Electrophysiology; D027341:Excitatory Amino Acid Transporter 1; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D008081:Liposomes; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D010957:Plasmids; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D027981:Symporters
chemical_list: D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D018076:DNA, Complementary; D027341:Excitatory Amino Acid Transporter 1; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D008081:Liposomes; C493398:Slc1a1 protein, rat; C493392:Slc1a3 protein, rat; D013438:Sulfhydryl Compounds; D027981:Symporters