AbstractGlutamate is the major excitatory neurotransmitter in the mammalian central nervous system(CNS), and is inactivated by cellular uptake, mostly catalyzed by glutamate (excitatory aminoacid) transporter subtype number 2 (EAAT2). EAAT2 protein is mostly found in astroglia(>80%), but there is also some in axon terminals (about 10 %). However, glia and nerveterminals in hippocampal slice preparations accumulate D-aspartate (D-Asp; an EAAT2substrate) with similar rates when incubated in vitro. This implies that there is an unexplainedmismatch between the distribution of EAAT2 transporter protein and the distribution oftransport activity. The main aim of the present study has been to find out if thedisproportionately high rate of uptake into terminals can be explained by differences in therelative rates of net uptake and of heteroexchange. To do this, glutamate transporters weresolubilized and reconstituted in artificial cell membranes (liposomes), and the liposomes weretested for their usefulness as a model: Uptake of external substrate required either internal K+or internal Na+ and glutamate, and liposomes that were preloaded with glutamate weresufficiently tight to keep most of the internal glutamate for the duration of the assay. Inagreement with the notion that the uptake is relatively robust to changes in the lipidenvironment, addition of polychlorinated biphenyls (PCBs) had no effect, while arachidonicacid inhibited exchange similar to net uptake. Uptake by K+-loaded liposomes was stimulatedby addition of a K+ ionophore (valinomycin), but the combination of permeant anions andvalinomycin appeared to cause rapid dissipation of driving forces. When the liposomes werestudied in the presence of valinomycin, K+-loaded liposomes performed better than liposomespreloaded with Na+ and glutamate, suggesting that net uptake is faster; at least atnon-saturating substrate concentrations (< 5 ìM). In conclusion, the findings may imply thatD-Asp uptake into terminals in hippocampal slice preparations is due to net uptake, and thatdirect uptake into terminals is more important than currently recognized.