Early postnatal inhibition of GLAST causes abnormalities of psychobehaviors and neuronal morphology in adult mice.

The importance of glutamate transporters in learning, memory, and emotion remains poorly understood; hence, in the present study, we investigated whether deficiency of pharmacological GLAST in neurodevelopmental processes affects cognitive and/or emotional behaviors in mice. The mice were injected with a glutamate transporter inhibitor, dl-threo-ß-benzyloxyaspartate (dl-TBOA), during the early postnatal period. At 8 weeks of age, they showed impairments in cognitive or emotional behaviors; dysfunction of glutamatergic neurotransmission (increased expressions of GLAST, GLT-1, or GFAP protein, and decreased ability of glutamate release) in the cortex or hippocampus; morphological changes (decreased cell size in the cortex and thickness of the pyramidal neuronal layer of the CA1 area in the hippocampus). Such behavioral and morphological changes were not observed in adult mice injected with dl-TBOA. These results suggest that GLAST plays an important role in the regulation of cognitive and emotional behaviors. Early postnatal glutamatergic facilitation by GLAST dysfunction leads to cognitive and emotional abnormalities due to neurodevelopmental abnormalities such as morphological changes.

Chronic optogenetic stimulation of Bergman glia leads to dysfunction of EAAT1 and Purkinje cell death, mimicking the events caused by expression of pathogenic ataxin-1.

Bergmann glia (BG) are highly specialized radial astrocytes of the cerebellar cortex, which play a key role in the uptake of synaptic glutamate via the excitatory amino acid transporter EAAT1. Multiple lines of evidence suggest that in cerebellar neurodegenerative diseases reactive BG has a negative impact on neuronal function and survival through compromised EAAT activity. A family of such diseases are those caused by expansion of CAG repeats in genes of the ataxin family, resulting in spinocerebellar ataxias (SCA). We investigated the contribution of BG to the pathogenesis of cerebellar neurodegeneration in a model of SCA1, which was induced by expression of a polyglutamine mutant of ataxin-1 (ATXN1[Q85]) in BG specifically. We compared the outcomes with a novel model where we triggered excitotoxicity by a chronic optogenetic activation of BG with channelrhodopsin-2 (ChR2). In both cases we detected evidence of reduced glutamate uptake manifested by prolongation of excitatory postsynaptic currents in Purkinje cells which is consistent with documented reduction of expression and/or function of EAAT1. In both models we detected astroglyosis and Purkinje cells atrophy. Finally, the same pattern was detected in a knock-in mouse which expresses a polyglutamine mutant ataxin-1 ATXN1[Q154] in a non-cell-selective manner. Our results suggest that ATXN1[Q85] and ChR2-induced insult targeted to BG closely mimics SCA1 pathology, where excessive glutamate signaling appears to be a common feature likely being an important contributor to cerebellar neurodegeneration.

Glutamate induced neonatal excitotoxicity modifies the expression level of EAAT1 (GLAST) and EAAT2 (GLT-1) proteins in various brain regions of the adult rat.

Glutamate-mediated excitatory synaptic signalling is primarily controlled by excitatory amino acid transporters (EAATs), such as EAAT1 and EAAT2, which are located mostly on astrocytes and, together, uptake more than 95 % of extracellular glutamate. Alterations in the functional expression levels of EAATs can lead to excessive extracellular glutamate accumulation, potentially triggering excitotoxicity and seizures, among other neurological disorders. Excitotoxicity induced in early developmental stages can lead to lasting changes in several neurotransmission systems, including the glutamatergic system, which could make the brain more susceptible to a second insult. In this study, the expression levels of EAAT1 (GLAST) and EAAT2 (GLT-1) proteins were assessed in the cerebral motor cortex (CMC), striatum, hippocampus and entorhinal cortex (EC) of male adult rats following the neonatal excitotoxic process triggered by monosodium glutamate (MSG)-treatment (4 g/kg of body weight at postnatal days 1,3,5 and 7, subcutaneously). Western blot analysis showed that neonatal MSG-treatment decreased EAAT1 expression levels in the CMC, striatum and hippocampus, while EAAT2 levels were increased in the striatum and EC and decreased in the CMC. Immunofluorescence staining confirmed the changes in EAAT1 and EAAT2 expression induced by neonatal MSG-treatment, which were accompanied by an increase in the glial fibrillary acidic protein (GFAP) immunofluorescence signalthat was particularly significant in the hippocampus. Our results show that a neonatal excitotoxic processes can induce lasting changes in the expression levels of EAAT1 and EAAT2 proteins and suggest that although astrogliosis occurs, glutamate uptake could be deficient, particularly in the CMC and hippocampus.

Astrocyte control of glutamatergic activity: Downstream effects on serotonergic function and emotional behavior.

Major depressive disorder (MDD) is a leading cause of disability worldwide, with a poorly known pathophysiology and sub-optimal treatment, based on serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors. We review existing theories on MDD, paying special attention to the role played by the ventral anterior cingulate cortex (vACC) or its rodent equivalent, infralimbic cortex (IL), which tightly control the activity of brainstem monoamine neurons (including raphe 5-HT neurons) via descending afferents. Further, astrocytes regulate excitatory synapse activity via glutamate reuptake through astrocytic transporters EAAT1 and EAAT2 (GLAST and GLT-1 in rodents), and alterations of astrocyte number/function have been reported in MDD patients and suicide victims. We recently assessed the impact of reducing GLAST/GLT-1 function in IL on emotional behavior and serotonergic function in rodents. The acute pharmacological blockade of GLT-1 with dihydrokainate (DHK) in rat IL evoked an antidepressant-like effect mediated by local AMPA-R activation and a subsequent enhancement of serotonergic function. No effects were produced by DHK microinfusion in prelimbic cortex (PrL). In the second model, a moderate small interfering RNAs (siRNA)-induced reduction of GLAST and GLT-1 expression in mouse IL markedly increased local glutamatergic neurotransmission and evoked a depressive-like phenotype (reversed by citalopram and ketamine), and reduced serotonergic function and BDNF expression in cortical/hippocampal areas. As for DHK, siRNA microinfusion in PrL did not evoke behavioral/neurochemical effects. Overall, both studies support a critical role of the astrocyte-neuron communication in the control of excitatory neurotransmission in IL, and subsequently, on emotional behavior, via the downstream associated changes on serotonergic function.

SLC1A3 contributes to L-asparaginase resistance in solid tumors.

L-asparaginase (ASNase) serves as an effective drug for adolescent acute lymphoblastic leukemia. However, many clinical trials indicated severe ASNase toxicity in patients with solid tumors, with resistant mechanisms not well understood. Here, we took a functional genetic approach and identified SLC1A3 as a novel contributor to ASNase resistance in cancer cells. In combination with ASNase, SLC1A3 inhibition caused cell cycle arrest or apoptosis, and myriads of metabolic vulnerabilities in tricarboxylic acid (TCA) cycle, urea cycle, nucleotides biosynthesis, energy production, redox homeostasis, and lipid biosynthesis. SLC1A3 is an aspartate and glutamate transporter, mainly expressed in brain tissues, but high expression levels were also observed in some tumor types. Here, we demonstrate that ASNase stimulates aspartate and glutamate consumptions, and their refilling through SLC1A3 promotes cancer cell proliferation. Lastly, in vivo experiments indicated that SLC1A3 expression promoted tumor development and metastasis while negating the suppressive effects of ASNase by fueling aspartate, glutamate, and glutamine metabolisms despite of asparagine shortage. Altogether, our findings identify a novel role for SLC1A3 in ASNase resistance and suggest that restrictive aspartate and glutamate uptake might improve ASNase efficacy with solid tumors.

Complete but not partial inhibition of glutamate transporters exacerbates cortical excitability in the R6/2 mouse model of Huntington's disease.

AIM: Deficient glutamate reuptake occurs in the cerebral cortex of Huntington's disease (HD) patients and murine models. Here, we examine the effects of partial or complete blockade of glutamate transporters on excitatory postsynaptic currents (EPSCs) of cortical pyramidal neurons (CPNs). METHODS: Whole-cell patch clamp recordings of CPNs in slices from symptomatic R6/2 mice and wild-type (WT) littermates were used to examine the effects of selective or concurrent inhibition of glutamate reuptake transporters. RESULTS: Selective inhibition of the glial glutamate transporter 1 (GLT-1) or the glutamate aspartate transporter (GLAST) produced slight decreases in decay time of evoked EPSCs in CPNs from WT and R6/2 mice with no significant differences between genotypes. In contrast, concurrent inhibition of both transporters with DL-TBOA induced a significant increase in area and decay time and this effect was significantly greater in R6/2 CPNs. Furthermore, full blockade also reduced spontaneous EPSC frequency and exacerbated epileptiform activity in CPNs from symptomatic R6/2 mice. CONCLUSIONS: R6/2 CPNs are more sensitive to glutamate accumulation during full inhibition of both glutamate transporters, and these neurons have homeostatic mechanisms to cope with inhibition of GLT-1 or GLAST by a mechanism that involves upregulation of either transporter when the other is deficient.

Chemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues As Novel Excitatory Amino Acid Transporter Inhibitors.

Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacological properties were characterized at the EAAT1-4 subtypes. The l- threo-3-substituted Asp derivatives 13a-e and 13g-k were nonsubstrate inhibitors, exhibiting pan activity at EAAT1-4 with IC50 values ranging from 0.49 to 15 µM. Comparisons between (dl- threo)-19a-c and (dl- erythro)-19a-c Asp analogues confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogues (3b-e) of l-TFB-TBOA (3a) were shown to be potent EAAT1-4 inhibitors, with IC50 values ranging from 5 to 530 nM. Hybridization of the nonselective EAAT inhibitor l-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compounds 8 and 9, respectively, which were nonselective EAAT inhibitors displaying considerably lower IC50 values at EAAT1-4 (11-140 nM) than those displayed by the respective parent molecules.

Valproic acid attenuates manganese-induced reduction in expression of GLT-1 and GLAST with concomitant changes in murine dopaminergic neurotoxicity.

Exposure to elevated levels of manganese (Mn) causes manganism, a neurological disorder with similar characteristics to those of Parkinson's disease (PD). Valproic acid (VPA), an antiepileptic, is known to inhibit histone deacetylases and exert neuroprotective effects in many experimental models of neurological disorders. In the present study, we investigated if VPA attenuated Mn-induced dopaminergic neurotoxicity and the possible mechanisms involved in VPA's neuroprotection, focusing on modulation of astrocytic glutamate transporters (glutamate aspartate transporter, GLAST and glutamate transporter 1, GLT-1) and histone acetylation in H4 astrocyte culture and mouse models. The results showed that VPA increased promoter activity, mRNA/protein levels of GLAST/GLT-1 and glutamate uptake, and reversed Mn-reduced GLAST/GLT-1 in in vitro astrocyte cultures. VPA also attenuated Mn-induced reduction of GLAST and GLT-1 mRNA/protein levels in midbrain and striatal regions of the mouse brain when VPA (200 mg/kg, i.p., daily, 21 d) was administered 30 min prior to Mn exposure (30 mg/kg, intranasal instillation, daily, 21 d). Importantly, VPA attenuated Mn-induced dopaminergic neuronal damage by reversing Mn-induced decrease of tyrosine hydroxylase (TH) mRNA/protein levels in the nigrostriatal regions. VPA also reversed Mn-induced reduction of histone acetylation in astrocytes as well as mouse brain tissue. Taken together, VPA exerts attenuation against Mn-induced decrease of astrocytic glutamate transporters parallel with reversing Mn-induced dopaminergic neurotoxicity and Mn-reduced histone acetylation. Our findings suggest that VPA could serve as a potential neuroprotectant against Mn neurotoxicity as well as other neurodegenerative diseases associated with excitotoxicity and impaired astrocytic glutamate transporters.

Photoswitchable Inhibitor of a Glutamate Transporter.

Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.

Structure and allosteric inhibition of excitatory amino acid transporter 1.

Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the molecular mechanisms underlying the function and pharmacology of human SLC1 transporters.

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation.

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl- ]int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na+ -K+ -2Cl- cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl- ]int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl- ]int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl- ]int . Other tested chloride channels or chloride transporters do not contribute to [Cl- ]int under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K+ -Cl- cotransporter change resting Bergmann glial [Cl- ]int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

ß-Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences.

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 ß-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC50 values ∼1 µM at all three subtypes, this elaborate structure-activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue 4y that displayed an IC50 of 0.8 µM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF3-phenyl analogue 4r was a potent selective EAAT2-inhibitor (IC50 = 2.8 µM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these ß-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles.

Identification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity Relationship Study.

Screening of a small compound library at the three excitatory amino acid transporter subtypes 1-3 (EAAT1-3) resulted in the identification of compound (Z)-4-chloro-3-(5-((3-(2-ethoxy-2-oxoethyl)-2,4-dioxothiazolidin-5-ylidene)methyl)furan-2-yl)benzoic acid (1a) that exhibited a distinct preference as an inhibitor at EAAT1 (IC50 20 µM) compared to EAAT2 and EAAT3 (IC50 > 300 µM). This prompted us to subject 1a to an elaborate structure-activity relationship study through the purchase and synthesis and subsequent pharmacological characterization of a total of 36 analogues. Although this effort did not result in analogues with substantially improved inhibitory potencies at EAAT1 compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently available pharmacological tools in the EAAT field but also substantiates the notion that EAAT ligands not derived from α-amino acids hold considerable potential in terms of subtype-selective modulation of the transporters.

Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes.

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-Benzylated l-threo-ß-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

Drosophila astrocytes cover specific territories of the CNS neuropil and are instructed to differentiate by Prospero, a key effector of Notch.

Astrocytes are crucial in the formation, fine-tuning, function and plasticity of neural circuits in the central nervous system. However, important questions remain about the mechanisms instructing astrocyte cell fate. We have studied astrogenesis in the ventral nerve cord of Drosophila larvae, where astrocytes exhibit remarkable morphological and molecular similarities to those in mammals. We reveal the births of larval astrocytes from a multipotent glial lineage, their allocation to reproducible positions, and their deployment of ramified arbors to cover specific neuropil territories to form a stereotyped astroglial map. Finally, we unraveled a molecular pathway for astrocyte differentiation in which the Ets protein Pointed and the Notch signaling pathway are required for astrogenesis; however, only Notch is sufficient to direct non-astrocytic progenitors toward astrocytic fate. We found that Prospero is a key effector of Notch in this process. Our data identify an instructive astrogenic program that acts as a binary switch to distinguish astrocytes from other glial cells.

New Insight into the Structure-Activity Relationships of the Selective Excitatory Amino Acid Transporter Subtype†1 (EAAT1) Inhibitors UCPH-101 and UCPH-102.

In the present study, we made further investigations on the structure-activity requirements of the selective excitatory amino acid transporter 1 (EAAT1) inhibitor, 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101), by exploring 15 different substituents (R(1) ) at the 7-position in combination with eight different substituents (R(2) ) at the 4-position. Among the 63 new analogues synthesized, we identified a number of compounds that unexpectedly displayed inhibitory activities at EAAT1 in light of understanding the structure-activity relationship (SAR) of this inhibitor class extracted from previous studies. Moreover, the nature of the R(1) and R(2) substituents were observed to contribute to the functional properties of the various analogues in additive and non-additive ways. Finally, separation of the four stereoisomers of analogue 14 g (2-amino-4-([1,1'-biphenyl]-4-yl)-3-cyano-7-isopropyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene) was carried out, and in agreement with a study of a related scaffold, the R configuration at C4 was found to be mandatory for inhibitory activity, while both the C7 diastereomers were found to be active as EAAT1 inhibitors. A study of the stereochemical stability of the four pure stereoisomers 14 g-A-D showed that epimerization takes places at C7 via a ring-opening, C-C bond rotation, ring-closing mechanism.

Bioavailability Studies and in†vitro Profiling of the Selective Excitatory Amino Acid Transporter Subtype†1 (EAAT1) Inhibitor UCPH-102.

Although the selective excitatory amino acid transporter subtype 1 (EAAT1) inhibitor UCPH-101 has become a standard pharmacological tool compound for in vitro and ex vivo studies in the EAAT research field, its inability to penetrate the blood-brain barrier makes it unsuitable for in vivo studies. In the present study, per os (p.o.) administration (40 mg kg(-1) ) of the closely related analogue UCPH-102 in rats yielded respective plasma and brain concentrations of 10.5 and 6.67 µm after 1 h. Three analogue series were designed and synthesized to improve the bioavailability profile of UCPH-102, but none displayed substantially improved properties in this respect. In vitro profiling of UCPH-102 (10 µm) at 51 central nervous system targets in radioligand binding assays strongly suggests that the compound is completely selective for EAAT1. Finally, in a rodent locomotor model, p.o. administration of UCPH-102 (20 mg kg(-1) ) did not induce acute effects or any visible changes in behavior.

Pharmacological inhibitions of glutamate transporters EAAT1 and EAAT2 compromise glutamate transport in photoreceptor to ON-bipolar cell synapses.

To maintain reliable signal transmission across a synapse, free synaptic neurotransmitters must be removed from the cleft in a timely manner. In the first visual synapse, this critical task is mainly undertaken by glutamate transporters (EAATs). Here we study the differential roles of the EAAT1, EAAT2 and EAAT5 subtypes in glutamate (GLU) uptake at the photoreceptor-to-depolarizing bipolar cell synapse in intact dark-adapted retina. Various doses of EAAT blockers and/or GLU were injected into the eye before the electroretinogram (ERG) was measured. Their effectiveness and potency in inhibiting the ERG b-wave were studied to determine their relative contributions to the GLU clearing activity at the synapse. The results showed that EAAT1 and EAAT2 plays different roles. Selectively blocking glial EAAT1 alone using UCPH101 inhibited the b-wave 2-24h following injection, suggesting a dominating role of EAAT1 in the overall GLU clearing capacity in the synaptic cleft. Selectively blocking EAAT2 on photoreceptor terminals had no significant effect on the b-wave, but increased the potency of exogenous GLU in inhibiting the b-wave. These suggest that EAAT2 play a secondary yet significant role in the GLU reuptake activity at the rod and the cone output synapses. Additionally, we have verified our electrophysiological findings with double-label immunohistochemistry, and extend the literature on the spatial distribution of EAAT2 splice variants in the mouse retina.

SLC1 glutamate transporters.

The plasma membrane transporters for the neurotransmitter glutamate belong to the solute carrier 1 family. They are secondary active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the cotransport of Na(+) ions and the countertransport of one K(+) in a step independent of the glutamate translocation step. Due to eletrogenicity of transport, the transmembrane potential can also act as a driving force. Glutamate transporters are expressed in many tissues, but are of particular importance in the brain, where they contribute to the termination of excitatory neurotransmission. Glutamate transporters can also run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. This review summarizes recent literature on the functional and biophysical properties, structure-function relationships, regulation, physiological significance, and pharmacology of glutamate transporters. Particular emphasis is on the insight from rapid kinetic and electrophysiological studies, transcriptional regulation of transporter expression, and reverse transport and its importance for pathophysiological glutamate release under ischemic conditions.

Links between L-glutamate transporters, Na+/K+-ATPase and cytoskeleton in astrocytes: evidence following inhibition with rottlerin.

Astrocytes are plastic cells that play key roles in brain physiology and pathology, including via their glutamate transporters, excitatory amino acid transporter (EAAT)1 and EAAT2, maintaining low extracellular glutamate concentrations and protecting against excitotoxic neuronal injury. Alterations in cell surface expression of EAATs and astrocytic cytoskeleton are important for regulating transporter activity. This study employed the actions of rottlerin, to interrogate the regulation of EAAT activity, expression and localization, and interfaces with Na(+)/K(+)-ATPase and astrocytic morphology. EAAT activity and expression were determined in primary cultures of mouse astrocytes in the presence of and after rottlerin removal, with or without trafficking inhibitors, using uptake ([(3)H]d-aspartate, (86)Rb(+)) and molecular analyses. Astrocytic morphology and EAAT localization were investigated using Western blotting and immunocytochemistry, in concert with image analysis of glial fibrillary acidic protein, F-actin and EAAT1/2. Rottlerin induced a time-dependent inhibition of glutamate transport (Vmax). Rapid changes in cytoskeletal arrangement were observed and immunoblotting revealed increases in EAAT2 total and cell surface expression, despite reduced EAAT activity. Rottlerin-induced inhibition was reversible and its rate was increased by monensin co-treatment. Rottlerin inhibited, while monensin stimulated Na(+)/K(+)-ATPase. Removal of rottlerin rapidly elevated Na(+)/K(+)-ATPase activity beyond control levels, while co-treatment with monensin failed to stimulate the Na(+)/K(+)-ATPase. These data reveal inhibition of EAAT activity by rottlerin is not associated with loss of EAATs at the cell surface, but rather linked to cytoskeletal rearrangement, and inhibition of the Na(+)/K(+)-ATPase. Rapid recovery of Na(+)/K(+)-ATPase activity, and subsequent restoration of glutamate uptake indicates that astrocytic morphology and EAAT activity are co-regulated by a tightly coupled, homeostatic relationship between l-glutamate uptake, the electrochemical gradient and the activity of the Na(+)/K(+)-ATPase.