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Results for “Excitatory Amino Acid Transporter 4”

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At least 19 recordsLinked to original sources

PubMed2005

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors.

Excitatory amino acid transporters (EAATs) are involved in regulating extracellular glutamate levels at synaptic regions in the CNS. EAAT1, 2, 3, and 5 have been found in the mammalian retina, but the presence of EAAT4 has remained controversial. Recently, we found a high level of EAAT4 mRNA in the human retina, and this observation lead us to examine whether EAAT4 was expressed in the mammalian retina. Immunoblotting studies showed the presence of EAAT4-immunoreactive proteins in human and mouse retinas, corresponding to EAAT4 monomers and dimers. Immunohistochemistry revealed that EAAT4 was localized in rod and cone photoreceptor outer segments in the human retina, and in the outer and inner segments of mouse and ground squirrel retinas. In no case was EAAT4 found in the outer plexiform layer or in any other layer in the retina. EAAT4 expression by photoreceptors was confirmed by immunoblotting a purified rod outer segment preparation, which showed the presence of a 50-kDa EAAT4-immunoreactive protein. In addition, the EAAT4-associated protein, GTRAP41, was found in the human, mouse, and squirrel retinas as well as in the rod outer segment preparation. Further immunocytochemical and co-immunoprecipitation experiments demonstrated that GTRAP41 was colocalized and interacted in vivo with EAAT4. Importantly, glutamate uptake and drug inhibition experiments showed that an EAAT4-like glutamate uptake system is present in the rod outer segments. Finally, we examined whether glutamate signaling mediated by EAAT4 can modulate rod outer segment phagocytosis by the retinal pigment epithelium. Results of the present study show that EAAT4 is present in the outer segments, a nonsynaptic region of photoreceptors, where it might provide a feedback mechanism for sensing extracellular glutamate or serve as an outer barrier to prevent glutamate from escaping from the retina.

Amino Acid Transport System X-AG↗

PubMed2004

Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina.

Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.

Amino Acid Transport System X-AG↗

PubMed2006

Polymorphisms in the canine glutamate transporter-1 gene: identification and variation among five dog breeds.

Excitatory amino acid transporters (EAATs) are important for terminating glutamatergic neurotransmission and protect central nervous system (CNS) neurons from glutamatergic excitotoxicity. We selected these genes as targets that may relate to canine behavioral traits. After screening four EAAT genes (glutamate transporter-1; GLT-1, excitatory amino acid transporter 4; EAAT4, excitatory amino acid carrier; EAAC1, glutamate/aspartate transporter; GLAST) for single nucleotide polymorphisms (SNPs), we identified two silent SNPs (C129T and T471C) in the GLT-1 gene. We genotyped 193 dogs of 5 breeds and found significant variation among breeds in these two SNPs in GLT-1. The C129T polymorphism was not observed in Malteses and Miniature Schnauzers. These results suggest that polymorphisms in the GLT-1 gene may be useful markers for examining how the genetic background relates to the behavioral traits of dogs.

Alleles↗

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↗

PubMed2005

Quantitative analysis of glutamate transporter mRNA expression in prefrontal and primary visual cortex in normal and schizophrenic brain.

Abnormalities of the glutamatergic system in schizophrenia have been identified in numerous studies, but little is known about the role of glutamate transporters and their messenger RNA (mRNA) expression. In addition, the abundances of the two major isoforms of human excitatory amino acid transporter 2 (EAAT2) or its rat ortholog, glutamate transporter 1, have never been compared in a quantitative manner. Using quantitative reverse transcription-polymerase chain reaction, we established that the expression of the EAAT1, EAAT2a, EAAT2b, and EAAT3 transcripts was not different in the dorsolateral prefrontal and primary visual cortices of persons with schizophrenia relative to matched controls. EAAT2a expression was about 25-fold and 10-fold higher than EAAT2b in human and rat brain, respectively. The data provided no evidence of an effect of antipsychotic medications on the mRNA expression of the glutamate transporters. However, because most of the schizophrenic subjects in the cohort had been treated with antipsychotics for many years, it is still possible that changes in transporter expression were masked by medication effects.

Actins↗

PubMed2004

Expression of glutamate transporter subtypes in cultured retinal pigment epithelial and retinoblastoma cells.

PURPOSE: Glutamate is the major excitatory neurotransmitter in the retina and glutamate uptake is essential for normal glutamate signalling. Retinal diseases may induce neurochemical changes which affect retinal cells including retinal pigment epithelium (RPE). The aim of the study was to investigate the expression of glutamate transporter subtypes in RPE and retinoblastoma cells and to clarify the effect of proliferation modulators on the levels of the expressed transporter in the RPE cell line. METHODS: Cultured pig RPE cells and two human RPE cell lines, D407 and ARPE-19, as well as the human retinoblastoma cell line Y79 were used. Glutamate transporter expression was evaluated with Western blot analysis and immunocytochemistry. RESULTS: The study revealed unexpected expression of neuronal glutamate transporter/chloride channel EAAT4 in these three cell lines, but not in cultured pig RPE cells, whereas another glutamate carrier, EAAC1, was present in all cell types utilized. Other transporter subtypes, GLT1, GLAST and EAAT5 were not found. Neither tamoxifen, known to inhibit both proliferation and glutamate uptake in RPE cells, nor retinoic acid nor insulin, also known to affect cell proliferation rates, were capable of changing the total levels of EAAT4 in APRE-19 cells. CONCLUSIONS: Neuronal glutamate transporter EAAC1 is expressed in RPE cells. The robust expression of EAAT4 in cell lines may reflect a role of EAAT4 in cell proliferation and migration. Unaltered steady-state expression of this carrier and chloride-channel protein hints at posttranslational mechanisms of regulation of EAAT4.

Amino Acid Transport System X-AG↗

PubMed2004

Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains: importance for excitatory amino acid transporter localization and function.

In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced Na(+)-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-beta-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na(+)-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function.

Amino Acid Transport System X-AG↗

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↗

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↗

PubMed2005

Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses.

Glutamate transporters are essential for terminating excitatory neurotransmission. Two distinct glutamate transporters, glutamate-aspartate transporter (GLAST) and excitatory amino acid transporter 4 (EAAT4), are expressed most abundantly in the molecular layer of the cerebellar cortex. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on Purkinje cell dendritic spines, whereas EAAT4 is concentrated on the extrasynaptic regions of Purkinje cell spine membranes. To clarify the functional significance of the coexistence of these transporters, we analyzed the kinetics of EPSCs in Purkinje cells of mice lacking either GLAST or EAAT4. There was no difference in the amplitude or the kinetics of the rising and initial decay phase of EPSCs evoked by stimulations of climbing fibers and parallel fibers between wild-type and EAAT4-deficient mice. However, long-lasting tail currents of the EPSCs appeared age dependently in most of Purkinje cells in EAAT4-deficient mice. These tail currents were never seen in mice lacking GLAST. In the GLAST-deficient mice, however, the application of cyclothiazide that reduces desensitization of AMPA receptors increased the peak amplitude of the EPSC and prolonged its decay more markedly than in both wild-type and EAAT4-deficient mice. The results indicate that these transporters play differential roles in the removal of synaptically released glutamate. GLAST contributes mainly to uptake of glutamate that floods out of the synaptic cleft at early times after transmitter release. In contrast, the main role of EAAT4 is to remove low concentrations of glutamate that escape from the uptake by glial transporters at late times and thus prevents the transmitter from spilling over to neighboring synapses.

Animals↗

PubMed2006

Intrinsic kinetics determine the time course of neuronal synaptic transporter currents.

Efficient clearance of synaptically released glutamate from the extracellular space is an absolute requirement for maintaining information processing in the central nervous system. In the cerebellum, clearance of glutamate relies on uptake by Bergmann glial cells and Purkinje cells (PCs). Uptake by PCs can be monitored by recording the synaptic transport current (STC) mediated by the PC-specific transporter excitatory amino acid transporter 4 (EAAT4). The slow time course of the PC STC has been used to argue that glutamate clearance is protracted. We find, however, that the time course of the STC is not affected by altering the amount of glutamate released at individual synapses or by partial transporter blockade, manipulations that would be expected to change the duration of the extracellular glutamate transient. Ion substitution experiments and kinetic modeling of the PC transporter current suggest that physiological levels of intracellular Na(+) and glutamate slow the cycling rate of transporters and thereby lengthen the time course of STCs. The model predicts that PC transporters bind glutamate quickly but that the actual cycling rate of EAAT4 in physiological conditions is slow; therefore, the STC reflects the intrinsic kinetics of the glutamate transporter, not the rate of glutamate clearance.

Animals↗

PubMed2006

Spatiotemporal analysis of purkinje cell degeneration relative to parasagittal expression domains in a model of neonatal viral infection.

Infection of newborn Lewis rats with Borna disease virus (neonatal Borna disease [NBD]) results in cerebellar damage without the cellular inflammation associated with infections in later life. Purkinje cell (PC) damage has been reported for several models of early-life viral infection, including NBD; however, the time course and distribution of PC pathology have not been investigated rigorously. This study examined the spatiotemporal relationship between PC death and zonal organization in NBD cerebella. Real-time PCR at postnatal day 28 (PND28) revealed decreased cerebellar levels of mRNAs encoding the glycolytic enzymes aldolase C (AldoC, also known as zebrin II) and phosphofructokinase C and the excitatory amino acid transporter 4 (EAAT4). Zebrin II and EAAT4 immunofluorescence analysis in PND21, PND28, PND42, and PND84 NBD rat cerebella revealed a complex pattern of PC degeneration. Early cell loss (PND28) was characterized by preferential apoptotic loss of zebrin II/EAAT4-negative PC subsets in the anterior vermis. Consistent with early preferential loss of zebrin II/EAAT4-negative PCs in the vermis, the densities of microglia and the Bergmann glial expression of metallothionein I/II and the hyaluronan receptor CD44 were higher in zebrin II/EAAT4-negative zones. In contrast, early loss in lateral cerebellar lobules did not reflect a similar discrimination between PC phenotypes. Patterns of vermal PC loss became more heterogeneous at PND42, with the loss of both zebrin II/EAAT4-negative and zebrin II/EAAT4-positive neurons. At PND84, zebrin II/EAAT4 patterning was abolished in the anterior cerebellum, with preferential PC survival in lobule X. Our investigation reveals regional discrimination between patterns of PC subset loss, defined by zebrin II/EAAT4 expression domains, following neonatal viral infection. These findings suggest a differential vulnerability of PC subsets during the early stages of virus-induced neurodegeneration.

Animals↗

PubMed1998

Molecular cloning and expression of the rat EAAT4 glutamate transporter subtype.

Amino Acid Transport System X-AG↗

PubMed2005

Patterned expression of Purkinje cell glutamate transporters controls synaptic plasticity.

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. If expressed at high enough densities, transporters can prevent activation of extrasynaptic receptors by rapidly lowering glutamate concentrations to insignificant levels. We find that synaptic activation of metabotropic glutamate receptors expressed by Purkinje cells is prevented in regions of rat cerebellum where the density of the glutamate transporter EAAT4 is high. The consequences of metabotropic receptor stimulation, including activation of a depolarizing conductance, cannabinoid-mediated presynaptic inhibition and long-term depression, are also limited in Purkinje cells expressing high levels of EAAT4. We conclude that neuronal uptake sites must be overwhelmed by glutamate to activate perisynaptic metabotropic glutamate receptors. Regional differences in glutamate transporter expression affect the degree of metabotropic glutamate receptor activation and therefore regulate synaptic plasticity.

Analysis of Variance↗

PubMed2005

Roles of glial glutamate transporters in shaping EPSCs at the climbing fiber-Purkinje cell synapses.

Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). Here we aimed to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporter, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors, DHK prolonged the decay time constant (tau(w)) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by approximately 80%, caused no change in tau(w) in WT in the absence of CTZ, whereas it prolonged tau(w) in the presence of CTZ. This prolonged value of tau(w) was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion ( approximately 20%) of functional transporters is preserved.

Amino Acid Transport System X-AG↗

PubMed1997

High affinity glutamate transporters: regulation of expression and activity.

L-Glutamic acid is a major excitatory neurotransmitter in the mammalian central nervous system. The termination of the glutamatergic transmission and the clearance of the excessive, neurotoxic concentrations of glutamate is ensured by a high affinity glutamate uptake system. Four homologous types of Na/K-dependent high affinity glutamate transporters, glutamate/aspartate transporter, glutamate transporter 1, excitatory amino acid carrier 1, and excitatory amino acid transporter 4, have recently been cloned and were assigned to a separate gene family, together with two neutral amino acid carriers, alanine/serine/cysteine transporter 1/serine/alanine/threonine transporter and adipocyte amino acid transporter. The genomic organization of these transporters is still under investigation. Very little is known about the nature of the factors and molecular mechanisms that regulate developmental, regional, and cell type-specific expression of the glutamate transporters and their aberrant functioning in neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and Alzheimer's disease). Some experimental conditions (e.g., ischemia, corticostriatal lesions, hyperosmolarity, culturing conditions) and several naturally occurring and synthetic compounds (e.g., glutamate receptor agonists, dopamine, alpha1- and beta-adrenergic agonists, cAMP, phorbol esters, arachidonic acid, nitric oxide, oxygen free radicals, amyloid beta-peptide, tumor necrosis factor-alpha, glucocorticosteroids, unidentified neuronal factors) affect the molecular expression and activity of glutamate transporters. Further elucidation of the molecular events that link epigenetic signals with transcriptional and post-transcriptional mechanisms (e.g., alternative splicing, translation and post-translational modifications) is crucial for the development of selective pharmacological tools and strategies interfering with the expression of the individual glutamate transporters.

Animals↗

PubMed2005

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism.

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.

Anions↗

PubMed1998

Expression of two glutamate transporters, GLAST and EAAT4, in the human cerebellum: their correlation in development and neonatal hypoxic-ischemic damage.

Normal development and hypoxic-ischemic changes of glutamate-aspartate transporters (GLAST) and excitatory amino acid transporter type 4 (EAAT4) were demonstrated in the human cerebellum. GLAST-immunoreactive Bergmann's glia and EAAT4-positive Purkinje cells showed a specific distribution and localization, and developed with age in the molecular and Purkinje cell layers. The dendrites and cell bodies of Purkinje cells, which showed EAAT4 immunoreactivity, were ensheathed by GLAST processes. In neonatal hypoxic-ischemic encephalopathy (HIE), GLAST immunoreactivity decreased in the molecular layer and increased in the inner granule cell layer at an early stage, and markedly increased in the Purkinje and inner granule cell layers at a late stage. EAAT4 immunoreactivity decreased with post-ischemic changes of Purkinje cells. GLAST reactivity changed more rapidly than EAAT4 in cases of HIE. These changes of GLAST and EAAT4 may be closely related to the vulnerability of Purkinje cells in hypoxia-ischemia. The glutamate transporter of Bergmann glia may play a more important role in the regulation of the extracellular glutamate concentration in hypoxia and/or ischemia.

ATP-Binding Cassette Transporters↗