Glutamate, the major excitatory neurotransmitter in brain, is almost exclusively intracellular due to the action of the glutamate transporters in the plasma membranes. To study the localization and properties of these proteins, we have raised antibodies specifically recognizing parts of the sequences of two cloned rat glutamate transporters, GLT-1 (Pines et al., 1992) and GLAST (Storck et al., 1992). On immunoblots the antibodies against GLT-1 label a broad heterogeneous band with maximum density at around 73 kDa, while the antibody against GLAST labels a similarly broad band at around 66 kDa in the cerebellum and a few kilodaltons lower in other brain regions. GLT-1 is expressed at the highest concentrations in the hippocampus, lateral septum, cerebral cortex, and striatum, while GLAST is preferentially expressed in the molecular layer of the cerebellum. However, both transporters are present throughout the brain, and have roughly parallel distributions in the cerebral hemispheres and brainstem. Preembedding light and electron microscopical immunocytochemistry shows that both GLT-1 and GLAST are restricted to astrocytes, which appear to express both proteins concomitantly, but in different proportions in different parts of the brain. Nerve terminal labeling was not observed. Both the amino and carboxyl terminals of GLT- 1 and GLAST are located intracellularly, indicating an even number of transmembrane segments. Antibodies against a synthetic peptide corresponding to amino acid residues 2–11 of the proposed sequence of GLT-1 recognize the native rat brain GLT-1 protein, confirming that the translation initiation site is at the first ATG.

Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the crosslinker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

Glutamate transport across the plasma membrane of neurons and glia is powered by the transmembrane electrochemical gradients for sodium, potassium, and pH, but there is controversy over the number of Na ϩ cotransported with glutamate. The stoichiometry of glutamate transporters is important because it determines a lower limit to the extracellular glutamate concentration, [glu] o , in both normal and pathological conditions. We used whole-cell clamping to study the stoichiometry of the glial transporter GLT-1, the most abundant glutamate transporter in the brain, expressed under control of the Tet-On system in a Chinese hamster ovary (CHO) cell line selected for low endogenous glutamate transport. After the induction of GLT-1 expression with doxycycline, glutamate evoked a Na

The reuptake of glutamate in neurons and astrocytes terminates excitatory signals and prevents the persistence of excitotoxic levels of glutamate in the synaptic cleft. This process is inhibited by oxygen radicals and hydrogen peroxide (H2O2). Here we show that another biological oxidant, peroxynitrite (ONOO-), formed by combination of superoxide (O2-) and nitric oxide (NO), potently inhibits glutamate uptake by purified or recombinant high affinity glutamate transporters reconstituted in liposomes. ONOO- reduces selectively the Vmax of transport; its action is fast (reaching > or = 90% within 20 s), dose-dependent (50% inhibition at 50 microM), persistent upon ONOO- (or by product) removal, and insensitive to the presence of the lipid antioxidant vitamin E in the liposomal membranes. Therefore, it likely depends on direct interaction of ONOO- with the glutamate transporters. Three distinct recombinant glutamate transporters from the rat brain, GLT1, GLAST, and EAAC1, exhibit identical sensitivity to ONOO . H2O2 also inhibits reconstituted transport, and its action matches that of ONOO- on all respects; however, this is observed only with 5-10 mM H202 and after prolonged exposure (10 min) in highly oxygenated buffer. NO, released from NO donors (up to 10 mM), does not modify reconstituted glutamate uptake, although in parallel conditions it promotes cGMP formation in synaptosomal cytosolic fraction. Overall, our results suggest that the glutamate transporters contain conserved sites in their structures conferring vulnerability to ONOO- and other oxidants.

Glutamine is involved in a variety of metabolic processes, including recycling of the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). The system N transporter SN1 mediates efflux as well as influx of glutamine in glial cells [Chaudhry et al. (1999), Cell, 99, 769-780]. We here report qualitative and quantitative data on SN1 protein expression in rat. The total tissue concentrations of SN1 in brain and in kidney are half and one-quarter, respectively, of that in liver, but the average concentration of SN1 could be higher in astrocytes than in hepatocytes. Light and electron microscopic immunocytochemistry shows that glutamatergic, GABAergic and, surprisingly, purely glycinergic boutons are ensheathed by astrocytic SN1 laden processes, indicating a role of glutamine in the production of all three rapid transmitters. A dedication of SN1 to neurotransmitter recycling is further supported by the lack of SN1 immunoreactivity in oligodendrocytes (cells rich in glutamine but without perisynaptic processes). All neuronal structures appear unlabelled implying that a different protein mediates glutamine uptake into nerve endings. In several regions, SN1 immunoreactivity is higher in association with GABAergic than glutamatergic synapses, in agreement with observations that exogenous glutamine increases output of transmitter glutamate but not GABA. Nerve terminals with low transmitter reuptake or high prevailing firing frequency are associated with high SN1 immunoreactivity in adjacent glia. Bergmann glia and certain other astroglia contain very low levels of SN1 immunoreactivity compared to most astroglia, including retinal Müller cells, indicating the possible existence of SN isoforms and alternative mechanisms for transmitter recycling.

Perturbations of the synaptic handling of glutamate have been implicated in the pathogenesis of brain damage after transient ischemia. Notably, the ischemic episode is associated with an increased extracellular level of glutamate and an impaired metabolism of this amino acid in glial cells. Glutamate uptake is reduced during ischemia due to breakdown of the electrochemical ion gradients across neuronal and glial membranes. We have investigated, in the rat hippocampus, whether an ischemic event additionally causes a reduced expression of the glial glutamate transporter GLT1 (Pines et al. 1992) in the postischemic phase. Quantitative immunoblotting, using antibodies recognizing GLT1, revealed a 20% decrease in the hippocampal contents of the transporter protein, 6 h after an ischemic period lasting 20 min induced by four vessel occlusion. In situ hybridization histochemistry with 35S labelled oligonucleotide probes or digoxigenin labelled riboprobes directed to GLT1 mRNA showed a decreased signal in the hippocampus, particularly in CA1. This reduction was more pronounced at 3 h than at 24 h after the ischemic event. We conclude that the levels of GLT1 mRNA and protein show a modest decrease in the postischemic phase. This could contribute to the delayed neuronal death typically seen in the hippocampal formation after transient ischemia.

Membrane-localized transporter proteins, expressed in both neurons and glial cells, are responsible for removal of extracellular glutamate in the mammalian CNS. The amounts and activities of these transporters may be under regulatory control. We demonstrate here that cortical lesions, which decrease striatal glutamate uptake in synaptosome-containing homogenates by approximately 50%, also decrease the striatal concentrations of the astrocytic glutamate transporter proteins, GLT-1 and GLAST by approximately 20-30%. Since GABA uptake activity was not decreased and glial fibrillary acidic protein was increased in the same samples, the lesion-induced losses of GLT-1 and GLAST were not caused by a general impairment of neuronal or glial function. The observed reduction in the two astrocytic glutamate transporters after corticostriatal nerve terminal degeneration indicates that their levels of expression are dependent on glutamatergic innervation.

A monoclonal antibody (9C4) shows that an [Na'+K']coupled glutamate transporter protein purified from rat brain runs electrophoretically as a wide band and is localized in neuroglial cell bodies and processes, but not in neurons. This confirms the findings with polyclonal antibodies [Neuroscience 51 (1992) 295-3101, and shows that the apparent heterogeneity in relative molecular mass is accounted for by a single antigenic epitope. By testing several synthetic peptides derived from the deduced amino acid sequences of two cloned rat brain glutamate transporters, the antigenic epitope was identified as residing within the peptide TQSVYDDTKNHRESNSNQC (residues 518-536) of one of these wature 360 (1992) 4644671.