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 exci sed from transfected human embryonic kidney 293 cells. In the presence of t he highly permeant anion SCN-, pulses of glutamate rapidly activated transi ent 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 a nion and stoichiometric currents displayed similar kinetics, suggesting tha t anion channel gating and stoichiometric charge movements are linked to ea rly transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectio nal influx or exchange. Analysis of deactivation and recovery kinetics in t hese two conditions suggests that, after binding, translocation of substrat e is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocyte s, distinguish it as an efficient sink for synaptically released glutamate.