L-glutamate Transport Research Articles

Characterization and pH dependence of L-glutamate transport in sarcolemmal vesicles from rat hearts

Transport of L-glutamate in purified sarcolemmal vesicles from rat heart was Na+ dependent and characterized by an apparent affinity value of 0.149 +/- 0.04 mM (n = 6). Na+ binding to the carrier was not very specific, since an inwardly directed K+ gradient could also increase vesicular uptake of L-glutamate. Uptake of L-glutamate into sarcolemmal vesicles was inhibited by L-aspartate and L-cysteinate. These characteristics of glutamate uptake are typical of transport via system XAG-. Acidification of either the intravesicular or the extravesicular space led to a significant increase in L-glutamate transport. This increase was more pronounced when the intravesicular space had been acidified. Thus intracellular acidosis of hypoxic or ischemic myocardium is directly linked to an increased uptake of glutamate.

L-glutamate uptake inhibitors may stimulate phosphoinositide hydrolysis in baby hamster kidney cells expressing mGluR1a via heteroexchange with L-glutamate without direct activation of mGluR1a.

The functional efficacies of inhibitors of L-glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. L-Serine-O-sulfate was an agonist at mGluR1a (EC50 = 70 microM), mGluR2 (EC50 = 25 microM), and mGluR4 (EC50 = 324 microM). L-Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, L-cysteine, and DL-threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC50 values of 43, 64, 463, and 488 microM, respectively, and displaced L-[3H]glutamate binding from membranes prepared from these cells with respective IC50 values of 48, 44, 79, and 139 microM. However, D-aspartate, L-trans-pyrrolidine-2,4-dicarboxylate, L-threo-3-hydroxyaspartate, and L-aspartate-beta-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC50 values of 73, 54, 57, and 430 microM) but did not displace L-[3H]glutamate binding. These compounds inhibited Na(+)-dependent L-glutamate uptake into baby hamster kidney cells with IC50 values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this L-glutamate transporter, was significantly attenuated in the presence of L-glutamate decarboxylase (EC 4.1.1.15) or L-alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mM L-trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 +/- 0.2 microM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous L-glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.

Characterization of Na +-dependent l-glutamate transport in canine erythrocytes

Characteristics of the high-affinity Na +-dependent l-glutamate transport system in canine erythrocytes were studied by using intact cells and resealed ghosts. The l-glutamate transport showed a precise dependence on extracellular Na + and intracellular K +. Kinetical analysis revealed that two Na + ions and one K + ion were involved in each l-glutamate transport cycle. The l-glutamate transport was inhibited most potently by threo-3-hydroxyaspartate and l-cysteinesulfinate (at 25 μM, 83% and 79% inhibition, respectively) and weakly by dihydrokainate and dl-α-aminoadipate (at 25 μM, 21% and 17% inhibition, respectively). From these stoichiometrical and pharmacological properties we concluded that the l-glutamate transport system in canine erythrocytes is a product of the l-glutamate transporter gene family and resembles a neuronal transporter rather than a glial one. l-Glutamate uptake was increased by internal, but not external, HCO 3 − when the internal and external anions of the erythrocytes were replaced by several other anions. Moreover, this enhancement was blocked by inhibition of carbonic anhydrase, which indicated that l-glutamate transport was at least partly dependent on HCO 3 − generated inside the cells. These observations indicate that anion countertransport is coupled to the high-affinity Na +- and K +-dependent l-glutamate transport in canine erythrocytes.

Cloning and characterization of a glutamate transporter cDNA from human brain and pancreas

l-Glutamate is the major excitatory neurotransmitter in the brain. Sufficient removal from the synaptic cleft after neurotransmission by the l-glutamate transport system is essential to prevent excitotoxicity and neurotoxicity. We isolated mRNA from human brain and pancreatic islet cells and screened for sequences of high homology to a previously characterized rat brain glutamate transporter. An isolated sequence (GLTR) shows a 87.5% and a 92.5% sequence similarity at the nucleotide and amino acid level, respectively, with a rat brain specific l-glutamate transporter but only a 65% homology to the recently cloned human glutamate/aspartate transporter. The human mRNA is differentially expressed in brain and to a lesser degree in pancreas and in fetal liver. The gene encoding for the newly identified cDNA is located on chromosome 5.

Functional properties and substrate specificity of the cloned L- glutamate/L-aspartate transporter GLAST-1 from rat brain expressed in Xenopus oocytes

The rat brain L-glutamate/L-aspartate transporter GLAST-1 is a member of a family of Na(+)-dependent high-affinity L-glutamate transporters proposed to be involved in the termination and modulation of excitatory neurotransmitter signals. Application of electrophysiological and radiotracer techniques on Xenopus oocytes expressing cloned GLAST-1 revealed that the apparent Km value of the transporter for L-glutamate and Na+ ions did not depend on voltage while the maximal transport rate increased with more negative potentials, indicative of a low-field access channel. The apparent Km value of the transporter for L-glutamate depends on the Na+ concentration, suggesting that substrate and ions are transported by GLAST-1 in a simultaneous manner. All of the L-glutamate uptake blockers tested either were substrates or did not affect the current induced by L-glutamate. The changes in the amplitude of the current induced by simultaneous application of two substrates can be interpreted by a competition for one binding site.

Counter‐transport of potassium by the glutamate uptake carrier in glial cells isolated from the tiger salamander retina.

1. To investigate the transport of potassium on the glutamate uptake carrier, the glutamate uptake current in isolated retinal Müller cells was monitored by whole-cell clamping, while measuring changes of potassium concentration outside the cells ([K+]o) with an ion-sensitive microelectrode. 2. Activating glutamate uptake led to an accumulation of potassium outside the cells, consistent with the hypothesis, based on less direct evidence, that the glutamate uptake carrier transports potassium out of the cell. 3. The glutamate-evoked rise of [K+]o showed the pharmacology and sodium dependence of glutamate uptake. 4. The rise in [K+]o was proportional to the uptake current flowing between 0 and -80 mV, implying that the ratio of K+ transported to charge transported by the uptake carrier is constant over this voltage range. The K+ to charge transport ratio was the same for uptake of D-aspartate and L-glutamate. 5. By clamping cells with pipettes containing solutions of different [K+], the dependence of the glutamate and aspartate uptake currents on intracellular [K+] was determined. L- and D-aspartate transport showed a smaller maximum uptake current (Imax), and a smaller apparent Michaelis constant (Km) for activation by intracellular K+, than did L-glutamate transport. The ratio of Imax to Km was the same for these three analogues, a result which can be predicted from simple models of the carrier's operation. 6. Fully activating glutamate uptake in Müller cells in the intact retina would produce a K+ load into the extracellular space of about 0.6 mM s-1. Suppression of glutamate release from photoreceptors by light will reduce K+ efflux from Müller cells in the outer retina; this may contribute to the light-evoked fall of [K+]o observed in the outer retina, and thus contribute to shaping the electroretinogram.

Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study.

The distributions of the mRNAs encoding the L-glutamate transporters GLT1 and GLAST were examined in the rat brain by in situ hybridization using 35S-labelled oligonucleotide probes. Probes directed to GLT1 produced dense labelling in the hippocampus, neocortex and neostriatum, and weak labelling in the cerebellum. In contrast, GLAST mRNA appeared to be greatly enriched in the cerebellum compared to other brain regions. While the intensity of the labelling for GLAST and GLT1 varied among different regions, their cellular distributions appeared to coincide inasmuch as both mRNAs were mainly expressed by glial cells. Labelling occurred, inter alia, in glial cells throughout the hippocampus, and in Golgi epithelial cells in the Purkinje cell layer of the cerebellum.

Formula omitted] and cis-1-aminocyclobutane-1, 3-dicarboxylate behave as transportable, competitive inhibitors of the high-affinity glutamate transporters

The ability of two conformationally restricted analogues of L-glutamate to function as nontransportable inhibitors of plasma membrane L-glutamate transport was investigated in primary cultures of cerebellar granule cells and cortical astrocytes. L -trans- Pyrrolidine-2,4- dicarboxylic acid ( L -trans PDC ) and cis-1-aminocyclobutane-1,3-dicarboxylic acid ( cis-ACBD) behaved as linear competitive inhibitors of the uptake of d-[ 3H]aspartate (used as a non-metabolizable analogue of L-glutamate) exhibiting K i values between 40 and 145 μm; L -trans- PDC being the more potent inhibitor in each preparation. However, both L -trans- PDC and cis-ACBD, over a concentration range of 1 μM-5 mM, dose-dependently stimulated the release of exogenously supplied d-[ 3H]aspartate from granule cells maintained in a continuous superfusion system. The stimulated release was independent of extracellular calcium ions; essentially superimposable dose-response profiles being obtained in the absence and presence of 1.3 mM CaCl 2 and yielding ec 50 values of 16–25 μM and 180–220 μM for L -trans- PDC and cis-ACBD, respectively. Stimulated release of d-[ 3H]aspartate was unaffected by either 300 μm d-(−)-2-amino-5-phosphonopentanoi acid [ d-APV; a selective antagonist of the N-methyl- d-aspartate (NMDA) receptor] or by 25 μM 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX; a selective antagonist of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor]. The release of d-[ 3H] aspartate following stimulation by either L -trans- PDC or cis-ACBD was however markedly attenuated following substitution in the superfusion medium of sodium ions by choline ions. Taken together, these results support an action of L -trans- PDC and cis-ACBD consistent with that of being competitive substrates rather than non-transportable blockers of the plasma membrane L-glutamate uptake system.

Pharmacological characterization of a cloned rat glutamate transporter (GluT-1)

Pharmacological properties of a cDNA clone encoding a high affinity, Na +-dependent l-glutamate transporter (GluT-1) were examined using Xenopus oocytes. l-[ 3H]glutamate transport was inhibited by the putative endogenous substrates l-aspartate ( K i = 65 μM) and l-glutamate ( K i = 70 μM). l-Homocysteate did not significantly inhibit high-affinity glutamate transport ( K i = 2.7 mM). Of the previously identified uptake inhibitors, DL-threo-β-hydroxyaspartate ( K i = 65 μM), l-cysteine sulfinate ( K i = 80 μM), β-glutamate ( K i = 475 μM) and l-aspartate-β-hydroxamate ( K i = 950 μM) inhibited l-glutamate uptake. The other l-glutamate uptake blockers examined, including dihydrokainate, l-α-aminoadipate and SITS, weakly inhibited uptake of l-glutamate with K i values in excess of 1 mM. These features of the inhibitor sensitivities of GluT-1 transport show that GluT-1 is less sensitive to these agents than previously characterized glutamate transporters in rat brain, suggesting that GluT-1 is a novel glutamate transporter with a unique pharmacologic profile.

Phosphorylation and modulation of brain glutamate transporters by protein kinase C.

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.

Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na(+)-independent transport of L-glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst-stage embryos. In contrast, the Na(+)-dependent transport of L-aspartate is not detectable in oocytes, but it is detectable in eight-cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x-c and X-AG, respectively. Here we report the detection of Na(+)-dependent L-aspartate transport, which increased as pig blastocysts developed, although Na(+)-dependent aspartate transport was not detected in pig oocytes. Mediated Na(+)-independent L-glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X-AG is similar in both the pig and the mouse, system x-c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species.

Pretreatment of rat brain slices with ouabain decreases chloride-dependent l-glutamate transport in synaptic membrane

Possible roles of (Na(+)-K+)-ATPase on regulation of Cl(-)-dependent L-glutamate (L-Glu) transport in rat synaptic membrane were examined. Pretreatment of rat cerebral slices with 20 micrograms/ml veratrine, 56 mM K+ and 20 microM monensin increased Cl(-)-dependent L-[3H]-Glu uptake into membrane vesicles prepared from the slices. Pretreatment with (Na(+)-K+)-ATPase inhibitors, 100 microM ouabain, 100 microM strophanthidin, 100 microM vanadate and K(+)-free medium, decreased the uptake by 25-30%, while 5 microM A23187 and 10 microM ruthenium red had no effect. Ouabain (100 microM) caused the maximal effect in 10-20 min of incubation. The effect of (Na(+)-K+)-ATPase inhibitors was reversible and characterized by a decrease in Vmax of the uptake. Addition of 5 microM ouabain abolished the increases in the uptake induced by either veratrine, 56 mM K+ or monensin. Dose-inhibition curve of ouabain on the increased Cl(-)-dependent L-[3H]-Glu uptake was bi-phasic; ouabain at 0.5-5 microM selectively diminished the stimulatory effect of veratrine and inhibited the basal uptake at higher concentrations. Veratrine had no effect on L-[3H]-Glu uptake in 5 microM strophanthidin containing or K(+)-free medium. These results suggest the involvement of (Na(+)-K+)-ATPase in the veratrine-induced increase in Cl(-)-dependent L-Glu transport in rat brain synaptic membranes.

Electrogenic L-glutamate uptake in Xenopus laevis oocytes expressing a cloned rat brain L-glutamate/L-aspartate transporter (GLAST-1)

The transport of L-glutamate into Xenopus laevis oocytes expressing the cloned L-glutamate/L-aspartate transporter (GLAST-1) from rat brain was studied using the voltage clamp technique. At a holding potential of -90 mV, a bath application of 100 microM L-glutamate induced an inward current (IGLAST) with an amplitude ranging from -5 to -30 nA. IGLAST did not require extracellular Ca2+, Mg2+, or Cl-, was larger at negative potentials, and did not reverse up to +80 mV. The current was dependent on external L-glutamate and Na+ with half-maximal amplitudes at 11 microM L-glutamate and 41 mM Na+. IGLAST saturated at 100 microM L-glutamate and 80 mM Na+. The Hill coefficient for Na+ and L-glutamate was 3.3 and 1.3, respectively, suggesting that 3 Na+ accompany the transport of 1 L-glutamate molecule. At low [Na+]o, IGLAST was enhanced by reducing [K+]o, an indication for the countertransport of K+. Reducing external pH from 7.4 to 6.0 did not change the amplitude of IGLAST. This argues against a glutamate/proton cotransport. The results provide evidence for GLAST-1 carrying out a high affinity, sodium-dependent L-glutamate transport with a proposed stoichiometry of 3 Na+, 1 L-glutamate-/1 K+.

Cloning and expression of a novel Na(+)-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters

A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT's apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

Na+ responses of two marine bacteria that did not appear to require Na+ for growth

Two Gram-negative heterotrophic marine bacterial strains had been reported not to require Na+ when grown on a chemically defined medium solidifed with purified agar and prepared without added Na+. When these strains were tested in a chemically defined liquid medium they required at least 3 mM Na + for growth. The agar used in the plating medium was found to contribute 3.3 mM Na+. Increasing the concentrations of Na+ in the liquid medium above 3 mM increased the rate and extent of growth of both organisms and decreased the lag periods. Optimal Na+ concentrations for growth varied from 100 to 500 mM depending on the organism and the carbon source in the medium. Na+ was also required for the transport of the carbon source into the cells. For the maximal rate of transport of L-glutamate, one organism required only 10 mM Na +, the other, 50 mM. For acetate and succinate transport the optimal Na+ concentrations varied from 30 to 200 mM depending on the substrate and the organism. When the initial rate of transport of glutamate into one of the organisms was plotted against Na+ concentration the reponse curve was sigmoid and a Hill plot of the data indicated that the transport protein may possess three binding sites for Na+. Evidence was obtained indicating that both organisms possess a Na+-stimulated NADH oxidase. The results indicate that there are marine bacteria that grow to a limited extent at appreciably lower concentrations of Na+ than have been realized previously and for these a much more definitive examination of the requirement for Na+ is necessary.Key words: marine bacteria, Na+ requirement, growth, membrane transport, NADH oxidase.

Subtypes of Sodium‐Dependent High‐Affinity L‐[3H]Glutamate Transport Activity: Pharmacologic Specificity and Regulation by Sodium and Potassium

Some data suggest that the sodium-dependent, high-affinity L-glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium-dependent transport of L-[3H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by < 65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were < 10-fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L-Glu, L-aspartate, and L-cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10-fold selective as inhibitors. These included beta-N-oxalyl-L-alpha,beta-diaminopropionate, alpha-methyl-DL-glutamate, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, and (2S,1'S,2'S,3'S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S,1'R,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG-II) was identified as the most potent (IC50 = 5.5 microM) and selective (60-100-fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L-homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L-CCG-II and L-homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.

Effect of toxin ofprymnesium patelliferumon neurotransmitter transport mechanisms: Development of a sensitive test method

A crude extract of the ichthyotoxic phytoflagellate Prymnesium patelliferum strongly inhibited the uptake of neurotransmitters into isolated nerve endings (synaptosomes) and synaptic vesicles. These systems were about 100 and 10 times more sensitive toward the algal toxin, respectively, than a standard hemolysis assay often used for testing the toxicity of Prymnesium spp. and other ichthyotoxic algae. Prymnesium patelliferum grown in phsophorus-deficient (-P) medium was about five times more active than when grown in phosphorus sufficient (+P) medium. The inhibition ratio between the high-affinity synaptosomal uptake of L-glutamate and gamma-aminobyturic acid (GABA) was 1/2.7 for the -P culture and 1/1.9 for the +P culture. The inhibition ratios for the low-affinity vesicular uptake of L-glutamate, GABA, and dopamine (DA) were 1/5.8/0.3 and 1/1.7/0.2, respectively. The synaptosomal transport of L-glutamate is a rapid, simple, and sensitive test method for toxicity determination of Prymnesium spp. and will be a useful tool in the further isolation and purification of the toxic principles of this and other related algae. It is suggested that the toxin interferes with ion channels or acts as an ionopore.

Erratum: Cloning and expression of a rat brain L-glutamate transporter

Nature 360, 464-467 (1992) THE third author's name was shown incorrectly in this letter. It should read Magnar Bj0ras, as above. In addition, Fig. 2b is not, as stated, a western blot but is an autoradiogram of an SDS-polyacrylamide gel electrophoresis gel.

Comparative analysis of sodium-dependent l-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex

Uptake of [ 3H] l-glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium- and potassium-dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. l- trans-PDC inhibited the synaptosomal glutamate transport 2–4-fold stronger than the astroglial uptake. The substrate analogues dl-threo-β-hydroxy-aspartate, dl-aspartate-β-hydroxamate, l-aspartate and d-aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas d-glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.

An [Na + + K +]coupled l-glutamate transporter purified from rat brain is located in glial cell processes

Polyclonal antibodies were generated against the major polypeptide (73,000 mol. wt) present in a highly purified preparation of the [Na + + K +]coupled l-glutamate transporter from rat brain. These antibodies were able to selectively immunoprecipitate the 73,000 mol. wt polypeptide as well as most of the l-glutamate transport activity—as assayed upon reconstitution—from crude detergent extracts of rat brain membranes. The immunoreactivity in the various fractions obtained during the purification procedure [Danbolt et al. (1990) Biochemistry 29, 6734–6740] closely correlated with the l-glutamate transport activity. Immunoblotting of a crude sodium dodecyl sulphate brain extract, separated by two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the antibodies recognized one 73,000 mol. wt protein species only. Deglycosylation of the protein gave a 10,000 reduction in molecular mass, but no reduction in immunoreactivity. These findings establish that the 73,000 mol. wt polypeptide represents the l-glutamate transporter or a subunit thereof. The antibodies also recognize a 73,000 mol. wt polypeptide and immunoprecipitate l-glutamate transport activity in extracts of brain plasma membranes from rabbit, pig, cow, cat and man. Using the antibodies, the immunocytochemical localization of the transporter was studied at the light and electron microscopic levels in rat central nervous system. In all regions examined (including cerebral cortex, caudatoputamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial cells rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared non-immunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us.