Isolation of current components and partial reaction cycles in the glial glutamate transporter EAAT2.
The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of L-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN(-), pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectional influx or exchange. Analysis of deactivation and recovery kinetics in these two conditions suggests that, after binding, translocation of substrate is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate.
Otis
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Department of Neurobiology, University of California, Los Angeles Medical Center, Los Angeles, California 90095-1763, USA. otist@ucla.edu
Kavanaugh
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D018528:ATP-Binding Cassette Transporters / Q000187:drug effects / Q000502:physiology*; D027322:Amino Acid Transport System X-AG; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D027342:Excitatory Amino Acid Transporter 2; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D006624:Hippocampus / Q000187:drug effects / Q000502:physiology; D006801:Humans; D016623:Ion Pumps / Q000187:drug effects / Q000502:physiology; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000502:physiology*; D014162:Transfection / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D027342:Excitatory Amino Acid Transporter 2; D016623:Ion Pumps; D017981:Receptors, Neurotransmitter; D018698:Glutamic Acid