Abstract
Abstract
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system
(CNS), and is inactivated by cellular uptake, mostly catalyzed by glutamate (excitatory amino
acid) 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 nerve
terminals in hippocampal slice preparations accumulate D-aspartate (D-Asp; an EAAT2
substrate) with similar rates when incubated in vitro. This implies that there is an unexplained
mismatch between the distribution of EAAT2 transporter protein and the distribution of
transport activity. The main aim of the present study has been to find out if the
disproportionately high rate of uptake into terminals can be explained by differences in the
relative rates of net uptake and of heteroexchange. To do this, glutamate transporters were
solubilized and reconstituted in artificial cell membranes (liposomes), and the liposomes were
tested 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 were
sufficiently tight to keep most of the internal glutamate for the duration of the assay. In
agreement with the notion that the uptake is relatively robust to changes in the lipid
environment, addition of polychlorinated biphenyls (PCBs) had no effect, while arachidonic
acid inhibited exchange similar to net uptake. Uptake by K+-loaded liposomes was stimulated
by addition of a K+ ionophore (valinomycin), but the combination of permeant anions and
valinomycin appeared to cause rapid dissipation of driving forces. When the liposomes were
studied in the presence of valinomycin, K+-loaded liposomes performed better than liposomes
preloaded with Na+ and glutamate, suggesting that net uptake is faster; at least at
non-saturating substrate concentrations (< 5 ìM). In conclusion, the findings may imply that
D-Asp uptake into terminals in hippocampal slice preparations is due to net uptake, and that
direct uptake into terminals is more important than currently recognized.