Excitatory Amino Acid Transporter EAAT2 Research Articles

Ubiquitination and the proteasome rather than caspase-3-mediated C-terminal cleavage are involved in the EAAT2 degradation by staurosporine-induced cellular stress.

Diminished glutamate (Glu) uptake via the excitatory amino acid transporter EAAT2, which normally accounts for ~90% of total forebrain EAAT activity, may contribute to neurodegeneration via Glu-mediated excitotoxicity. C-terminal cleavage by caspase-3 (C3) was reported to mediate EAAT2 inactivation and down-regulation in the context of neurodegeneration. For a detailed analysis of C3-dependent EAAT2 degradation, we employed A172 glioblastoma as well as hippocampal HT22 cells and murine astrocytes over-expressing VSV-G-tagged EAAT2 constructs. C3 activation was induced by staurosporine (STR). In HT22 cells, STR-induced C3 activation-induced rapid EAAT2 protein degradation. The mutation of asparagine 504 to aspartate (D504N), which should inactivate the putative C3 cleavage site, increased EAAT2 activity in A172 cells. In contrast, the D504N mutation did not protect EAAT2 protein against STR-induced degradation in HT22 cells, whereas inhibition of caspases, ubiquitination and the proteasome did. Similar results were obtained in astrocytes. Phylogenetic analysis showed that C-terminal ubiquitin acceptor sites-but not the putative C3 cleavage site-exhibit a high degree of conservation. Moreover, C-terminal truncation mimicking C3 cleavage increased rather than decreased EAAT2 activity and stability as well as protected EAAT2 against STR-induced ubiquitination-dependent degradation. We conclude that cellular stress associated with endogenous C3 activation degrades EAAT2 via a pathway involving ubiquitination and the proteasome but not direct C3-mediated cleavage. In addition, C3 cleavage of EAAT2, described to occur in other models, is unlikely to inactivate EAAT2. However, mutation of the highly conserved D504 within the putative C3 cleavage site increases EAAT2 activity via an unknown mechanism.

Adaptations induced by lacking maternal Kinin B1 receptor and maternal swimming during pregnancy in the placenta and in fetal growth

Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [3H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10 min of cocaine administration and lasted for up to 30 min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.

Astrocytes and Glutamine Synthetase in Epileptogenesis.

The cellular, molecular, and metabolic mechanisms that underlie the development of mesial temporal lobe epilepsy are incompletely understood. Here we review the role of astrocytes in epilepsy development (a.k.a. epileptogenesis), particularly astrocyte pathologies related to: aquaporin 4, the inwardly rectifying potassium channel Kir4.1, monocarboxylate transporters MCT1 and MCT2, excitatory amino acid transporters EAAT1 and EAAT2, and glutamine synthetase. We propose that inhibition, dysfunction or loss of astrocytic glutamine synthetase is an important causative factor for some epilepsies, particularly mesial temporal lobe epilepsy and glioblastoma-associated epilepsy. We postulate that the regulatory mechanisms of glutamine synthetase as well as the downstream effects of glutamine synthetase dysfunction, represent attractive, new targets for antiepileptogenic interventions. Currently, no antiepileptogenic therapies are available for human use. The discovery of such interventions is important as it will fundamentally change the way we approach epilepsy by preventing the disease from ever becoming manifest after an epileptogenic insult to the brain.

Regulation of Ion Channels, Cellular Carriers and Na(+)/K(+)/ATPase by Janus Kinase 3

Janus kinase-3 (JAK3), a tyrosine kinase, is expressed in a variety of tissues, including the brain and is involved in the signaling of cytokine receptors. JAK3 participates in numerous functions, such as cell survival and proliferation, neuroprotection, apoptosis and the cellular response to hypoxia and ischemia-reperfusion. This kinase further contributes to the signaling of hematopoietic cell cytokine receptors, activation of dendritic cells, maturation, and immune suppression as well as to cell volume regulation. Recently, JAK3 has been demonstrated to be an important regulator of transport processes across the plasma membrane. Either directly or indirectly JAK3 affects the expression of transport proteins, including various ion channels, a number of cellular carriers and the Na+/K+ pump. More specifically, JAK3 is involved in the regulation of various potassium, sodium, and chloride ion channels, a wide variety of Na+-coupled cellular carriers including the high-affinity Na+ coupled glucose transporter SGLT1, the excitatory amino acid transporters EAAT1, EAAT2, EAAT3 and EAAT4, the peptide transporters PepT1 and PepT2, CreaT1 and theNa+/K+-ATPase. Via these transporters this kinase plays a role in various physiological and pathophysiological processes. Additional research is needed to investigate the effects of JAK3 on other cellular transporters and the underlying mechanisms.

Inhibitory effect of yokukansan on the decrease in the hippocampal excitatory amino acid transporter EAAT2 in stress-maladaptive mice

Chronic stress is widely recognized as a risk factor for the development of major depression and anxiety disorders. Recently, we reported that yokukansan (YKS), a traditional Japanese herbal medicine, alleviated emotional abnormality in stress-maladaptive mice. The aim of the present study was to examine the effect of YKS on the expression of excitatory amino acid transporter (EAAT) 1–4 in the prefrontal cortex and hippocampus in stress-maladaptive mice. Mice were chronically exposed to inadaptable stress, i.e. repeated restraint stress for 240 min/day for 14 days. After the final exposure to stress, brains of mice were rapidly removed and the hippocampus and prefrontal cortex were dissected. Expressions of EAAT1-4 and glial fibrillary acidic protein (GFAP), a marker of astrocytes, in the brain tissues were analyzed by western blotting. Western blot analysis revealed that the expression level of EAAT2 was specifically decreased in the hippocampus of stress-maladaptive mice while there were no changes in the level of GFAP, and this change was inhibited by chronic treatment with YKS. In contrast, no changes were observed in the levels of EAAT1, EAAT3 or EAAT4 in stress-maladaptive mice. These results suggest that YKS may protect against the decrease in hippocampal EAAT2 expression induced by stress maladaptation, and this may contribute, at least in part, to the improvement of emotional abnormality.

EAAT2 and the Molecular Signature of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapid and fatal neurodegenerative disease, primarily affecting upper and lower motor neurons. It is an extremely heterogeneous disease in both cause and symptom development, and its mechanisms of pathogenesis remain largely unknown. Excitotoxicity, a process caused by excessive glutamate signaling, is believed to play a substantial role, however. Excessive glutamate release, changes in postsynaptic glutamate receptors, and reduction of functional astrocytic glutamate transporters contribute to excitotoxicity in ALS. Here, we explore the roles of each, with a particular emphasis on glutamate transporters and attempts to increase them as therapy for ALS. Screening strategies have been employed to find compounds that increase the functional excitatory amino acid transporter EAAT2 (GLT1), which is responsible for the vast majority of glutamate clearance. One such compound, ceftriaxone, was recently tested in clinical trials but unfortunately did not modify disease course, though its effect on EAAT2 expression in patients was not measured.

Single exposure to cocaine impairs aspartate uptake in the pre-frontal cortex via dopamine D1-receptor dependent mechanisms

Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [3H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.

Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR). The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3) or EAAT2 (SLC1A2) was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (I_EAAT). EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable I_EAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. I_EAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal I_EAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.

Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates L-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for L-aspartate over D-aspartate and L-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.

Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1.

SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by (T233E)SPAK and (T185E)OSR1, but not by (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2.

Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho

Background/Aims: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required for inhibition of 1,25(OH)₂D₃ formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. At least in part by exerting β-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2), Na⁺ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability. Methods: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM)-induced inward current (I_Glu) taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Results: I_Glu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes I_Glu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers. Conclusion: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation.

Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: A light and electron microscopic study

The process of glutamate release, activity, and reuptake involves the astrocyte, the presynaptic and postsynaptic neurons. Glutamate is released into the synapse and may occupy and activate receptors on both neurons and astrocytes. Glutamate is rapidly removed from the synapse by a family of plasma membrane excitatory amino acid transporters (EAATs), also localized to neurons and astrocytes. The purpose of the present study was to examine EAAT labeling in the postmortem human cortex at the light and electron microscopic (EM) levels. The postmortem prefrontal cortex was processed for EAAT1 and EAAT2 immunohistochemistry. At the light microscopic level, EAAT1 and EAAT2 labeling was found in both gray and white matter. Most cellular labeling was in small cells which were morphologically similar to glia. In addition, EAAT1-labeled neurons were scattered throughout, some of which were pyramidal in shape. At the EM level, EAAT1 and EAAT2 labeling was found in astrocytic soma and processes surrounding capillaries. EAAT labeling was also found in small astrocytic processes adjacent to axon terminals forming asymmetric (glutamatergic) synapses. While EAAT2 labeling was most prevalent in astrocytic processes, EAAT1 labeling was also present in neuronal processes including the soma, axons, and dendritic spines. Expression of EAAT1 protein on neurons may be due to the hypoxia associated with the postmortem interval, and requires further confirmation. The localization of EAATs on the astrocytic plasma membrane and adjacent to excitatory synapses is consistent with the function of facilitating glutamate reuptake and limiting glutamate spillover. Establishment that EAAT1 and EAAT2 can be measured at the EM level in human postmortem tissues will permit testing of hypotheses related to these molecules in diseases lacking analogous animal models.

Vesicular glutamate transporter and cognition in stroke

Vascular dementia (VaD) accounts for approximately 15%-20% of all dementias, but the relationship of progressive cognitive impairment to neurochemical changes is poorly understood. We have therefore investigated glutamatergic synaptic markers in VaD. We used homogenates prepared from gray matter from 2 neocortical regions (Brodmann area [BA] 9 and BA 20) and Western blotting to determine the concentrations of key components of the glutamatergic neurotransmitter system, vesicular glutamate transporter 1 (VGLUT1) and excitatory amino acid transporter EAAT2 (GLT-1), and the ubiquitous synaptic protein, synaptophysin, in 73 individuals-48 patients with cerebrovascular disease with and without dementia, 10 patients with AD, and 15 controls-in a case-control design. VGLUT1 concentrations in BA 20 and BA 9 were correlated with CAMCOG total (Rs 0.525, p = 0.018, n = 20; Rs 0.560, p = 0.002, n = 27) and CAMCOG memory scores (Rs 0.616, p = 0.004, n = 20; Rs 0.675, p = 0.000, n = 27). VGLUT1 concentration in BA 9 differed between the different dementia groups and the stroke no dementia group (1-way analysis of variance F = 6.69, p = 0.001 and Bonferroni p < 0.01 in each case), with subjects with stroke who did not develop dementia exhibiting the highest mean value for VGLUT1. These data suggest that loss of glutamatergic synapses is a feature of VaD and Alzheimer disease but the preservation of synapses, in particular glutamatergic synapses, in the frontal cortex against the temporal cortex plays a role in sustaining cognition and protecting against dementia following a stroke.

Ethanol Induces Expression of the Glutamate Transporters EAAT1 and EAAT2 in Organotypic Cortical Slice Cultures

Exposure of the developing brain to ethanol disposes the fetus to fetal alcohol syndrome and causes a number of changes in several neurochemical systems. In particular, the glutamatergic system is affected by ethanol. Thus, increased sensitivity of glutamate receptors and enhanced transmembrane transport of glutamate were found in primary astrocyte cultures. However, in these in vitro studies, changes in the expression of glutamate transporters were not detected. To further characterize the influence of chronic ethanol exposure on the developing brain, we assessed the transcriptional and translational regulation of glutamate transporters in a less artificial in vitro system. We exposed postnatal rat cortical organotypic slice cultures to ethanol (100 mM) for 4 and 8 days. Expression of the excitatory amino acid transporters EAAT1 and EAAT2 was analyzed in comparison to untreated controls with semiquantitative reverse transcriptase-polymerase chain reaction. In addition, changes in protein expression were detected by Western blotting. In ethanol-exposed cortical slices, we observed more prominent fiber outgrowth and significantly increased gene expression (EAAT1: +35%, p = 0.029; EAAT2: +251%, p = 0.015). These findings could be verified on the protein level, because Western blot analysis revealed significantly higher levels of EAAT1 (+76%; p = 0.008) and EAAT2 (+104%; p = 0.018) in ethanol-treated slices compared with controls. Our results are in concert with earlier studies describing the induction of glutamate transport by ethanol. Enhanced expression of EAAT1 and EAAT2 after ethanol exposure can be viewed as a maladaptive process that disposes the developing brain to fetal alcohol syndrome.

Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [ 3 H ]- d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR ® Membrane Potential (FMP) assay. The K m and K i values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K i values obtained in the [ 3 H ]- d-aspartate assay ( r 2 values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics of the cell lines in the FMP assay were in good agreement with previous findings in electrophysiology studies of the transporters. The FMP assay was capable of distinguishing between substrates and non-substrate inhibitors and to discriminate between “full” and “partial” substrates at the transporters. Taking advantage of the prolific nature of the FMP assay, interactions of the EAATs with substrates and inhibitors were studied in some detail. This is the first report of a high throughput screening assay for EAATs. We propose that the assay will be of great use in future studies of the transporters. Although conventional electrophysiology set-ups might be superior in terms of studying sophisticated kinetic aspects of the uptake process, the FMP assay enables the collection of considerable amounts of highly reproducible data with relatively little labor. Furthermore, considering that the number of EAAT ligands presently available is limited, and that almost all of these are characterized by low potency and a low degree of subtype selectivity, future screening of compound libraries at the EAAT-cell lines in the FMP assay could help identify structurally and pharmacologically novel ligands for the transporters.

Excitatory Amino Acid Transporter EAAT‐2 in Tangle‐bearing Neurons in Alzheimer's Disease

The excitatory amino acid transporter EAAT-2 is physiologically expressed in astrocytes. This study demonstrates that distinct subclasses of neurons exhibited EAAT-2 immunoreactivity in cases with Alzheimer's disease (AD). EAAT-2 was identified in the following types of neurons: Cortical pyramidal cells, fascia dentata granule cells, neurons of the basal nucleus of Meynert, the substantia nigra, the paraventricular nucleus of the hypothalamus, oral and central raphe nuclei, locus coeruleus, parabrachial nucleus, and neurons of the reticular formation of the brain stem. All EAAT-2-positive neurons displayed cytoskeletal abnormalities with abnormal tau-protein and often showed condensed and shrunken nuclei. None of the control cases without AD-related pathology showed EAAT-2-immunoreactive neurons. These results indicate that AD-related neurodegeneration is associated with the expression of the glutamate transporter EAAT-2 in altered neurons. Since an aberrant expression of EAAT-1 in neurons has recently been described, the finding of a neuronal expression of EAAT-2 strongly supports the hypothesis that abnormalities in glutamate transport play an important role in the pathogenesis of AD.

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes.

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.

Isolation of Current Components and Partial Reaction Cycles in the Glial Glutamate Transporter EAAT2

The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of L-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN(-), pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectional influx or exchange. Analysis of deactivation and recovery kinetics in these two conditions suggests that, after binding, translocation of substrate is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate.

5'-heterogeneity of the human excitatory amino acid transporter cDNA EAAT2 (GLT-1).

TWO novel transcripts of the human excitatory amino acid transporter EAAT2 (GLT-1) were cloned using PCR cloning techniques. Comparative sequence analysis of the 5'-untranslated region (UTR) of five EAAT2 transcripts led to the definition of five putative 5'-untranslated exons that are combined in a variable manner. Alternative splicing is a likely mechanism for the 5' complexity of the EAAT2 cDNA. The potential functional meaning of this finding such as differential expression of the EAAT2 transcripts in the central nervous system remains to be demonstrated.