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Results for “Excitatory Amino Acid Transporter 3”

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PubMed1997

Cellular and synaptic localization of the neuronal glutamate transporters excitatory amino acid transporter 3 and 4.

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. The present study was an investigation of the protein expression and cellular localization of EAAT4 in human and rat brain, and comparison with another neuronal glutamate transporter, EAAT3 (rabbit excitatory amino acid carrier 1; EAAC1). Regional immunoblot analysis of EAAT4, using a monospecific oligopeptide (carboxy-terminal) affinity-purified polyclonal antibody, revealed that the protein was restricted to the central nervous system. The EAAT4 protein was largely expressed in cerebellum, with a much lower expression in hippocampus, neocortex, striatum, brain stem and thalamus. Immunohistochemical studies showed intense EAAT4 immunoreactivity in the human and rat cerebellar Purkinje cells with a somatodendritic localization. Other brain regions including neocortex, hippocampus, striatum showed faint neuropil staining of EAAT4. Immunogold localization identified EAAT4 protein at plasma membranes of Purkinje cell dendrites and spines. In the hippocampus and neocortex, EAAT4 immunoreactivity was found mainly at small calibre dendrites. Rarely, EAAT4 immunoreactivity was found in astrocytic cell processes of forebrain. In the cerebellum, EAAT4 localization partly overlapped with the neuronal localization of EAAT3 (EAAC1). Immunoreactivity for EAAT3 was enriched in the somatodendritic compartment of the Purkinje cells like EAAT4, but EAAT3 was also found in Purkinje cell axons and in boutons in deep cerebellar nuclei, as well as in granular cells and stellate cells. Our results indicate that EAAT4 protein is largely localized to cerebellar cortex and lower levels of EAAT4 protein are present in forebrain by immunoblot and immunohistochemistry. Both neuronal glutamate transporter EAAT3 (EAAC1) and EAAT4 are located at somatodendritic compartment of Purkinje cells, and probably contribute to glutamate re-uptake mechanisms at Purkinje cell synapses.

Aged↗

PubMed2002

A novel sorting motif in the glutamate transporter excitatory amino acid transporter 3 directs its targeting in Madin-Darby canine kidney cells and hippocampal neurons.

The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and localized to the dendritic compartment in hippocampal neurons, where it is clustered adjacent to postsynaptic sites. In this study, we analyzed the sequences in EAAT3 that are responsible for its polarized localization in Madin-Darby canine kidney (MDCK) cells and neurons. Confocal microscopy and cell surface biotinylation assays demonstrated that deletion of the EAAT3 C terminus or replacement of the C terminus of EAAT3 with the analogous region in EAAT1 eliminated apical localization in MDCK cells. The C terminus of EAAT3 was sufficient to redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to the apical surface. Using alanine substitution mutants, we identified a short peptide motif in the cytoplasmic C-terminal region of EAAT3 that directs its apical localization in MDCK cells. Mutation of this sequence also impairs dendritic targeting of EAAT3 in hippocampal neurons but does not interfere with the clustering of EAAT3 on dendritic spines and filopodia. These data provide the first evidence that an identical cytoplasmic motif can direct apical targeting in epithelia and somatodendritic targeting in neurons. Moreover, our results demonstrate that the two fundamental features of the localization of EAAT3 in neurons, its restriction to the somatodendritic domain and its clustering near postsynaptic sites, are mediated by distinct molecular mechanisms.

Amino Acid Motifs↗

PubMed2005

Specificity of antibodies: unexpected cross-reactivity of antibodies directed against the excitatory amino acid transporter 3 (EAAT3).

UNLABELLED: Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479-498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. CONCLUSIONS: Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because cross-reactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.

Amino Acid Sequence↗

PubMed1996

Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1).

Several subtypes of sodium-dependent high affinity (SDHA) glutamate transporters have been pharmacologically differentiated in brain tissue. Recently, four distinct cDNAs (EAAC1, GLT1, GLAST, and EAAT4) encoding Na+-dependent glutamate transporters have been isolated, but the properties of some of these transporters do not fully match the properties of transport observed in brain tissue or astrocyte-enriched cultures. The purpose of the current investigation was to determine whether the pharmacological properties of EAAC1 parallel those observed in cortical or cerebellar synaptosomes, C6 glioma, or primary astrocyte-enriched cultures. EAAC1 cRNA was expressed in Xenopus oocytes, an expression system with no detectable endogenous Na+-dependent glutamate transport activity. EAAC1-mediated glutamate transport was >98% Na+ dependent, and the transport was saturable and consistent with a single site. Glutamate transport activates in EAAC1-injected oocytes and C6 glioma have similar Km values for glutamate (Km = 15-24 microM) and Na+ (apparent Km = 35-50mM), and these values markedly differ from those observed in rat synaptosomes (glutamate, Km = 1-5 microM; Na+, Km = 13-20 mM). Several excitatory amino acid analogues were tested as inhibitors of L-[3H] glutamate transport in oocytes expressing EAAC1 cRNA. The potencies of several compounds for inhibition of EAAC1-mediated transport differed from those previously observed in cerebellar synaptosomes and astrocyte-enriched cultures. Although EAAC1-mediated transport and cortical synaptosomal transport have similar pharmacological profiles, five excitatory amino acid analogues were > or= 3-fold more potent as inhibitors of transport into cortical synaptosomes than of transport into EAAC1-injected oocytes. For example, L-trans-pyrrolidine-2,4-dicarboxylate was approximately 5-fold more potent in cortical synaptosomes, and dihydrokainate was approximately 10-fold more potent in cortical synaptosomes than in EAAC1-injected oocytes. In contrast, all of the compounds examined inhibit transport observed in C6 glioma wtih potencies similar to that observed in oocytes injected with EAAC1 cRNA. Consistent with these data, C6 glioma expressed EAAC1- but not GLT1- and GLAST-like immunoreactivity. Although this immunoreactivity migrated as proteins of slightly different molecular masses in each system, treatment with N-glycosidase F shifted all proteins to a molecular mass consistent with that predicted from the cDNA sequence. In cortical synaptosomes, EAAC1-, GLT1-, and GLAST-like immunoreactives were apparent. These results indicate that (i) EAAC1 but not GLAST or GLT1 transporters are expressed in C6 glioma, (ii) synaptosomes contain a heterogeneous population of transporters, (iii) EAAC1 does not account for the pharmacology previously observed in cortical synaptosomes, and (iv) based on the pharmacology and tissue distribution of EAAC1, GLT1, GLAST, and EAAT4, it appears that there are additional glutamate transporter subtypes.

Amino Acid Transport System X-AG↗

PubMed2005

Isoflurane induces a protein kinase C alpha-dependent increase in cell-surface protein level and activity of glutamate transporter type 3.

Glutamate transporters regulate extracellular concentrations of glutamate, an excitatory neurotransmitter in the central nervous system. We have shown that the commonly used anesthetic isoflurane increased the activity of glutamate transporter type 3 (excitatory amino acid transporter 3, EAAT3) possibly via a protein kinase C (PKC)-dependent pathway. In this study, we showed that isoflurane induced a time- and concentration-dependent redistribution of EAAT3 to the cell membrane in C6 glioma cells. This redistribution was inhibited by staurosporine, a pan PKC inhibitor, or by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go6976) at a concentration that selectively inhibits conventional PKC isozymes (PKC alpha, -beta, and -gamma). This isoflurane-induced EAAT3 redistribution was also blocked when the expression of PKC alpha but not PKC beta proteins was down-regulated by the respective antisense oligonucleotides. The isoflurane-induced increase of glutamate uptake by EAAT3 was abolished by the down-regulation of PKC alpha expression. Immunoprecipitation with an anti-EAAT3 antibody pulled down more PKC alpha in cells exposed to isoflurane than in control cells. Isoflurane also increased the phosphorylated EAAT3 and the redistribution of PKC alpha to the particulate fraction of cells. Consistent with the results in C6 cells, isoflurane also increased EAAT3 cell-surface expression and enhanced the association of PKC alpha with EAAT3 in rat hippocampal synaptosomes. Our results suggest that the isoflurane-induced increase in EAAT3 activity requires an increased amount of EAAT3 protein in the plasma membrane. These effects are PKC alpha-dependent and may rely on the formation of an EAAT3-PKC alpha complex. Together, these results suggest an important mechanism for the regulation of glutamate transporter functions and expand our understanding of isoflurane pharmacology at cellular and molecular levels.

Amino Acid Transport System X-AG↗

PubMed2001

Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins.

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1-5). GLAST and GLT-1 are localized primarily in astrocytes, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9-11) and EAAT5 (ref 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4--the glutamate transporter expressed predominately in the cerebellum--or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

Amino Acid Sequence↗

PubMed2001

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems.

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na(+)-dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.

2-Amino-5-phosphonovalerate↗

PubMed2005

Functional expression of particular isoforms of excitatory amino acid transporters by rodent cartilage.

In the present study, we have attempted to demonstrate functional expression by the rodent cartilage of particular isoforms of excitatory amino acid transporters (EAATs) essentially required for central glutamatergic signal termination. Constitutive expression of mRNA was shown for the first time with the neuronal EAAT subtype excitatory amino acid carrier-1 (EAAC1), in addition to glial subtypes such as glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), in rat costal chondrocytes cultured for 7-21 days on reverse transcription polymerase chain reaction (RT-PCR). Western blotting analysis confirmed the expression of corresponding proteins for both GLAST and GLT-1 in cultured chondrocytes. The accumulation of [(3)H]glutamate (Glu) occurred in a temperature- and sodium-dependent manner with biochemical and pharmacological profiles similar to those seen for brain EAATs in chondrocytes cultured for 7 days, while [(3)H]Glu accumulation consisted of a single component with a K(m) of 39.1+/-2.3 microM and a V(max) of 1320+/-120pmol/mg protein/min, respectively. In organotypic cultured metatarsals isolated before vascularization from embryonic mice, where cells underwent maturational development from resting to proliferating, prehypertrophic, hypertrophic and calcified chondrocytes in a progressive order of cellular differentiation, moreover, mRNA expression was seen for GLAST, GLT-1 and EAAT4 but not for EAAC1 subtypes. Immunohistochemical analysis revealed distribution profiles different from each other with GLAST, GLT-1 and EAAT4 isoforms in sections of cultured metatarsals and isolated tibiae. These results suggest that extracellular Glu could be cleared up into intracellular locations through particular glial and/or neuronal EAAT isoforms functionally expressed by the rodent cartilage.

Amino Acid Transport System X-AG↗

PubMed2006

The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia.

Maintaining a low extracellular glutamate concentration in the central nervous system is important for terminating synaptic transmission and preventing excitotoxic cell death. The stoichiometry of the most abundant glutamate transporter, GLT-1, predicts that a very low glutamate concentration, approximately 2 nM, should be reached in the absence of glutamate release, yet microdialysis measurements give a value of approximately 1 microM. If other glutamate transporters had a different stoichiometry, the predicted minimum glutamate concentration could be higher, for example if those transporters were driven by the cotransport of 2 Na+ (rather than of 3 Na+ as for GLT-1). Here we investigated the ionic stoichiometry of the glutamate transporter GLAST, which is the major glutamate transporter expressed in the retina and cerebellum, is expressed in other adult brain areas at a lower level than GLT-1, and is present throughout the brain early in development when expression of GLT-1 is low. Glutamate transport by GLAST was found to be driven, as for GLT-1, by the cotransport of 3 Na+ and 1 H+ and the counter-transport of 1 K+, suggesting that the minimum extracellular glutamate concentration should be similar during development and in the adult brain. A less powerful accumulation of glutamate by GLAST than by GLT-1 cannot be used to explain the high glutamate concentration measured by microdialysis.

Amino Acid Transport System X-AG↗

PubMed2001

Molecular identification of high-affinity glutamate transporters in sheep and cattle forestomach, intestine, liver, kidney, and pancreas.

Glutamate metabolism is essential to support many facets of metabolism. The objective of this study was to determine the tissue distribution of glutamate transporters known to support the tissue metabolism of glutamate. The expression of proteins capable of high-affinity glutamate transport (system X-(AG)) by epithelia isolated from the rumen, omasum, duodenum, jejunum, ileum, cecum, and colon and homogenates of liver, kidney, and pancreatic tissues from wethers (n = 4; BW = 28.4 +/- 8.4 kg) and steers (n = 3; BW = 426 +/- 32.3 kg) fed forage-based diets was evaluated by immunoblot analysis. Proteins EAAC1 (62, 93 kDa) and GLT-1 (142, 188, >202 kDa) were expressed by every tissue examined. In contrast, GLAST1 (140 kDa) was expressed only by the pancreas, and EAAT4 (67 kDa) was detected only in sheep brain. To corroborate protein expression data, the presence and size of transporter mRNA in ileal, liver, and pancreatic homogenates were evaluated by Northern analysis. GLAST1 mRNA (2.4, 4.3 kb) was detected only in the pancreas, whereas EAAC1 (2.2, 2.8 kb) and GLT-1 (12.1 kb) mRNA transcripts were detected in all three tissues. The expression of EAAT4 and GLT-1 mRNA was confirmed by reverse transcriptase-polymerase chain reaction analyses. Sequencing of the resulting partial-length ovine GLT-1 cDNA revealed 100% identity with the rat homolog. Overall, these data demonstrate that sheep and cattle share the same pattern of system X-(AG) transporter expression, which differed among tissues and transporter isoforms. Accordingly, these data provide the fundamental knowledge to initiate research that determines whether the expression of high-affinity glutamate transporters by ruminants is sensitive to ontogenic and(or) dietary regulation.

ATP-Binding Cassette Transporters↗

PubMed2007

Association study of polymorphisms in the glutamate transporter genes SLC1A1, SLC1A3, and SLC1A6 with schizophrenia.

Based on the glutamatergic dysfunction hypothesis for schizophrenia pathogenesis, we have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. We report here association studies of schizophrenia with three glutamate transporter genes SLC1A1, SLC1A3, and SLC1A6 encoding the glutamate transporters EAAT3, EAAT1, and EAAT4, respectively. We initially performed the screening of the total 25 single nucleotide polymorphisms (SNPs) distributed in the three gene regions using 100 out of 400 Japanese cases and 100 out of 420 Japanese controls. After controlling the false discovery rate (FDR) at level 0.05, we observed significant associations of schizophrenia with a genotype of SNP4 (rs2097837, P = 0.007) and with haplotypes of SNP2-SNP5 (P = 7.5 x 10(-5)) and SNP3-SNP5 (P = 9.0 x 10(-4)) in the SLC1A6 region. The haplotype of SNP2-SNP5 of SLC1A6 even showed marginally significant association with the disease in the full-size sample (400 cases and 420 controls, P = 0.031). We concluded that at least one susceptibility locus for schizophrenia may be located within or nearby SLC1A6, whereas SLC1A1 and SLC1A3 are unlikely to be major susceptibility genes for schizophrenia in the Japanese population.

Adult↗

PubMed2003

Neuronal glutamate transporter EAAT4 is expressed in astrocytes.

High-affinity excitatory amino acid transporters (EAATs) are essential to terminate glutamatergic neurotransmission and to prevent excitotoxicity. To date, five distinct EAATs have been cloned from animal and human tissues: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5. EAAT1 and EAAT2 are commonly known as glial glutamate transporters, whereas EAAT3, EAAT4, and EAAT5 are neuronal. EAAT4 is largely expressed in cerebellar Purkinje cells. In this study, using immunohistochemistry and Western blotting, we found that EAAT4-like immunoreactivity (ir) is enriched in the spinal cord and forebrain. Double-labeled fluorescent immunostaining and confocal image analysis indicated that EAAT4-like ir colocalizes with an astrocytic marker, glial fibrillary acidic protein (GFAP). The astrocytic localization of EAAT4 was further confirmed in astrocyte cultures by double-labeled fluorescent immunocytochemistry and Western blotting. Reverse transcriptase-polymerase chain reaction analysis demonstrated mRNA expression of EAAT4 in astrocyte cultures. Sequencing confirmed the specificity of the amplified fragment. These results demonstrate that EAAT4 is expressed in astrocytes. This astrocytic localization of neuronal EAAT4 may reveal a new function of EAAT4 in the central nervous system.

Amino Acid Transport System X-AG↗

PubMed2004

Glutamate transporter expression and function in human glial progenitors.

Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by glutamate transporters. Five glutamate transporters in the human brain (EAAT1-5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early-differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP-derived astrocytes in the presence of bone morphogenic protein (BMP-4). This corresponds to a significant increase in the glutamate transport capacity of these cells. EAAT2, the transporter responsible for the bulk of glutamate transport in the adult brain, is not expressed as a full-length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early-differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal glutamate transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in glutamate transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.

Amino Acid Transport System X-AG↗

PubMed2004

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex.

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.

Amino Acid Transport System X-AG↗

PubMed1998

Glutamate uptake in Purkinje cells in rat cerebellar slices.

We have described how whole-cell clamping of neurons in brain slices has allowed a characterization of postsynaptic transporters, probably a mixture of EAAC1 and EAAT4, in cerebellar Purkinje cells. Similar experiments have been carried out on transporters (mainly GLAST) in cerebellar Bergmann glia, and have revealed an uptake current occurring as these carriers remove glutamate released at the parallel fiber synapses. As more transporters are cloned and their regulation is characterized in heterologous expression systems, it will be increasingly important to use methods similar to those outlined above to investigate to what extent the behavior of the carriers is similar in situ in the nervous system.

Amino Acid Transport System X-AG↗

PubMed2002

Striatal excitatory amino acid transporter transcript expression in schizophrenia, bipolar disorder, and major depressive disorder.

Because abnormalities of glutamatergic neurotransmission in psychiatric illness are likely not limited to glutamate receptor expression, we investigated expression of excitatory amino acid transporters (EAATs) in the striatum. The EAATs, normally expressed in both glia (EAAT1 and EAAT2) and neurons (EAAT3 and EAAT4), have previously been implicated in Huntington's disease, amyotrophic lateral sclerosis, and schizophrenia. In this study, we investigated striatal expression of transcripts encoding EAATs in tissue from mood disordered and schizophrenic subjects. With probes designed for the human EAAT1, EAAT2, EAAT3, and EAAT4 transcripts, we performed in situ hybridization and detected decreased expression of EAAT3 and EAAT4 transcripts in the striatum in bipolar disorder. We also detected decreased EAAT3 transcript expression in schizophrenia and decreased EAAT4 transcript expression in major depressive disorder. These results suggest that changes in striatal transporter mRNA expression are restricted to neuronal EAATs and extend the body of evidence implicating abnormal glutamatergic neurotransmission in schizophrenia and mood disorders.

Adult↗

PubMed2002

Selective loss of expression of glutamate GluR2/R3 receptor subunits in cerebellar tissue from a patient with olivopontocerebellar atrophy.

Expression of the mRNAs encoding the astrocytic (EAAT1, EAAT2) and neuronal (EAAT3, EAAT4) excitatory amino acid transporters and the AMPA-type glutamate receptor subunits GluR2 and GluR3 was investigated in postmortem cerebellar extracts from a patient with olivopontocerebellar atrophy (OPCA) and in material from three age-matched controls. Decreased expression in the steady state level of EAAT4 mRNA in the OPCA sample was correlated with the selective loss of Purkinje cells. Neuropathological evaluation revealed reactive gliosis and concomitantly increased expression of the mRNA encoding astrocytic glial fibrillary acidic protein (GFAP). Expression of the mRNAs encoding the AMPA receptor subunits GluR2 and GluR3 subunits was found to be decreased in OPCA suggesting that excitotoxic mechanism could play a role in the pathogenesis of the selective neuronal cell death in this disorder.

Aged↗

PubMed2003

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings.

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Amino Acid Transport System X-AG↗