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.

Within the adult mammalian dentate gyrus (DG) of the hippocampus, glutamate stimulates neural stem cell (NSC) self-renewing proliferation, providing a link between adult neurogenesis and local circuit activity. Here, we show that glutamate-induced self-renewal of adult DG NSCs requires glutamate transport via excitatory amino acid transporter 1 (EAAT1) to stimulate lipogenesis. Loss of EAAT1 prevented glutamate-induced self-renewing proliferation of NSCs in vitro and in vivo, with little role evident for canonical glutamate receptors. Transcriptomics and further pathway manipulation revealed that glutamate simulation of NSCs relied on EAAT1 transport-stimulated lipogenesis. Our findings demonstrate a critical, direct role for EAAT1 in stimulating NSCs to support neurogenesis in adulthood, thereby providing insights into a non-canonical mechanism by which NSCs sense and respond to their niche.

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. We hypothesize that a protein similar to EAAT1 found in brain may function as a glutamate transporter in cardiac mitochondria. Rat heart total RNA was screened by reverse transcriptase-polymerase chain reaction with an array of primer pairs derived from the rat brain EAAT1 cDNA sequence, yielding a 3786-bp cDNA comprising a 1638-bp open reading frame identical to rat brain EAAT1 with flanking 5'- and 3'-untranslated regions. Northern blot analysis confirmed a 4-kb mRNA product in rat heart and brain, with greater abundance in brain. A protein of the predicted approximate 60-kD size was recognized in myocardial lysates by an anti-EAAT1 polyclonal antibody produced against an amino-terminal peptide from human EAAT1. The protein enriched in rat heart mitochondria by immunoblot, co-localized with the mitochondrial protein cytochrome c by immunohistochemistry, and further localized to the inner mitochondrial membrane upon digitonin fractionation of the mitochondria. In myocytes overexpressing EAAT1, activity of the malate/aspartate shuttle increased by 33% compared to non-transfected cells (P = 0.004). These data indicate that EAAT1 is expressed in myocardial mitochondria, and functions in the malate/aspartate shuttle, suggesting a role for EAAT1 in myocardial glutamate metabolism.

It is routinely stated in the literature that Excitatory Amino Acid Transporter 5 (EAAT5) is a retina-specific glutamate transporter. EAAT5 is expressed by retinal photoreceptors and bipolar cells, where it serves as a slow transporter and as an inhibitory glutamate receptor, the latter role is due to the gating of a large chloride conductance. The dogma of an exclusively retinal distribution has arisen because Northern blot analyses have previously shown only modest hybridisation in non-retinal tissues. Others have re-interpreted this as indicating that EAAT5 was only present in retinal tissues. However, this view appears to be erroneous; recent evidence demonstrating abundant expression of EAAT5 in rat testis prompted us to re-examine this dogma. A new antibody was developed to an intracellular loop region of rat EAAT5. This new tool, in concert with RT-PCR and sequencing, demonstrated that EAAT5 is widely distributed at the mRNA and protein levels in many non-nervous tissues including liver, kidney, intestine, heart, lung, and skeletal muscle. We conclude that EAAT5 is a widely distributed protein. Whether it functions in all locations as a glutamate transporter, or mainly as a glutamate-gated chloride conductance, remains to be determined.

Excitatory amino acid transporters (EAATs) remove glutamate from the synaptic cleft. In the retina, EAAT1 and EAAT2 are considered the major glutamate transporters. However, it has not yet been possible to determine how EAAT5 shapes the retinal light responses because of the lack of a selective EAAT5 blocker or EAAT5 knock-out (KO) animal model. In this study, EAAT5 was found to be expressed in a punctate manner close to release sites of glutamatergic synapses in the mouse retina. Light responses from retinae of wild-type (WT) and of a newly generated model with a targeted deletion of EAAT5 (EAAT5-/-) were recorded in vitro using multielectrode arrays (MEAs). Flicker resolution was considerably lower in EAAT5-/- retinae than in WT retinae. The close proximity to the glutamate release site makes EAAT5 an ideal tool to improve temporal information processing in the retina by controlling information transfer at glutamatergic synapses.

The eukaryotic endocytic pathway regulates protein levels available at the plasma membrane by recycling them into specific endosomal compartments. ARFGAP1 is a component of the coat protein I (COPI) complex but it also plays a role in promoting adapter protein-2 (AP-2) mediated endocytosis. The excitatory amino acid transporter-3 (EAAT3) mediates the reuptake of glutamate from the synaptic cleft to achieve rapid termination of synaptic transmission at glutamatergic synapses. In this study, we identified two interacting proteins of EAAT3 by mass spectrometry (MS) ARFGAP1 and ARF6. We explored the role of ARFGAP1 and ARF6 in the endocytosis of EAAT3. Our data revealed that ARFGAP1 plays a role in the recycling of EAAT3, by utilizing its GTPase activating protein (GAP) activity and ARF6 acting as the substrate. ARFGAP1 promotes cargo sorting of EAAT3 via a single phenylalanine residue (F508) located at the C-terminus of the transporter. ARFGAP1-promoted AP-2 dependent endocytosis is abolished upon neutralizing F508. We utilized a heterologous expression system to identify an additional motif in the C-terminus of EAAT3 that regulates its endocytosis. Impairment in endocytosis did not affect somatodendritic targeting in cultured hippocampal neurons. Our findings support a model where endocytosis of EAAT3 is a multifactorial event regulated by ARFGAP1, occurring via the C-terminus of the transporter, and is the first study to examine the role of ARFGAP1 in the endocytosis of a transport protein.

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

Background: Excitatory amino acid transporter 2 encoded by SLC1A2 is responsible for approximately 90% of glutamate uptake. Glycine transporter 1, encoded by SLC6A9, is responsible for maintaining a low concentration of the N-methyl-D-aspartate receptor (NMDAR) co-agonist - glycine in the synaptic cleft, suggesting its participation in the development of the NMDARs hypofunction described in schizophrenia. Aim: The aim of this study was to evaluate whether the functional polymorphism-181 A/C (rs4354668) of the SLC1A2 and the rs2486001 (IVS3+411 G/A) in the SLC6A9 are involved in schizophrenia development and its clinical picture in the Polish population. Methods: The study group consisted of 393 unrelated Caucasian patients (157 [39.9%] females and 236 [60.1%] males; mean age 41±12) diagnosed with schizophrenia according to the DSM-5, and 462 healthy controls. The results of the Positive and Negative Syndrome Scale (PANSS) were presented in the five-dimensional model. Polymorphisms of SLC1A2 and SLC6A9 were genotyped with the use of PCR-RFLP assay. Results: There were no statistically significant differences in the frequency of genotypes and alleles between the patients and controls for SLC1A2 and SLC6A9 polymorphisms in either the entire sample or after stratification according to gender. In the haplotype analysis, men with CA haplotype had more than 1.5 higher risk to develop schizophrenia than women (OR=1.63 [95% CI=1.17-2.27, p<0.05]). The influence of gender, genotypes of both analyzed polymorphisms and gender x genotype interactions on individual dimensions of the PANSS scale has not been observed. Also, there was no association of either polymorphism with suicide attempts. Conclusion: The results of the present study did not indicate an association of polymorphism-181 A/C (rs4354668) in SLC1A2 and rs2486001 in SLC6A9 with onset of schizophrenia and its psychopathology in a Polish population.

The present study aimed to compare the expression levels of excitatory amino acid transporters (EAATs) and growth status of piglets weaned at 10‑20 days after birth with suckling piglets. A total of 40 hybrid piglets (Landrace x Large White x Duroc) born to 40 different sows, with similar body weight were selected for the present study. They were randomly divided into two groups (n=20 per group): Control group (suckling piglets) and experimental group (weaned piglets, reared in isolation). The experiment lasted for 10 days. At the end of the experiment, 12 piglets were randomly selected from each group and the jejunum and the ileum were collected in order to determine excitatory amino acid carrier 1 (EAAC1) expression levels and free amino acid content. The present study determined that early weaning significantly reduced EAAC1 gene and protein (57 and 73 kDa) expression levels and glutamate transporter associate protein 3‑18 (GTRAP3‑18; 50 kDa) in the jejunum and the ileum compared with the suckling group (P<0.05). Weaning led to an increased content of free glutamic acid (Glu) and total amino acids in the jejunum; however, content of free Glu and total amino acids in the ileum was significantly reduced (P<0.05). Early weaning reduced the expression of EAAC1 and GTRAP3‑18, which was possibly due to the amino acid absorption and transport disorder in the small intestine due to the Glu deficiency.

The glutamatergic dysfunction hypothesis of schizophrenia suggests that genes involved in glutametergic transmission are candidates for schizophrenic susceptibility genes. We have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. In this study we report an association study of the excitatory amino acid transporter 2 gene, SLC1A2 with schizophrenia.

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.

Hypoxia is a critical pathological element in many human diseases, including ischemic stroke, myocardial infarction, and solid tumors. Of particular significance and interest of ours are the cellular and molecular mechanisms that underlie susceptibility or tolerance to low O2. Previous studies have demonstrated that Notch signaling pathway regulates hypoxia tolerance in both Drosophila melanogaster and humans. However, the mechanisms mediating Notch-conferred hypoxia tolerance are largely unknown. In this study, we delineate the evolutionarily conserved mechanisms underlying this hypoxia tolerant phenotype. We determined the role of a group of conserved genes that were obtained from a comparative genomic analysis of hypoxia-tolerant D.melanogaster populations and human highlanders living at the high-altitude regions of the world (Tibetans, Ethiopians, and Andeans). We developed a novel dual-UAS/Gal4 system that allows us to activate Notch signaling in the Eaat1-positive glial cells, which remarkably enhances hypoxia tolerance in D.melanogaster, and, simultaneously, knock down a candidate gene in the same set of glial cells. Using this system, we discovered that the interactions between Notch signaling and bnl (fibroblast growth factor), croc (forkhead transcription factor C), or Mkk4 (mitogen-activated protein kinase kinase 4) are important for hypoxia tolerance, at least in part, through regulating neuronal development and survival under hypoxic conditions. Becausethese genetic mechanisms are evolutionarily conserved, this group of genes may serve as novel targets for developing therapeutic strategies and have a strong potential to be translated to humans to treat/prevent hypoxia-related diseases.

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.

Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.

The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.

The glial excitatory amino acid transporter 2 (EAAT2) mediates a majority of glutamate re-uptake in human CNS and, consequently, is associated with a variety of signaling and pathological processes. While our understanding of the function, mechanism and structure of this integral membrane protein is increasing, little if any mass spectrometric (MS) data is available for any of the EAATs specifically, and for only a few mammalian plasma membrane transporters in general. A protocol to express and purify functional EAAT2 in sufficient quantities to carry out MS-based peptide mapping as needed to study ligand-transporter interactions is described. A 6xHIS epitope was incorporated into the N-terminus of human EAAT2. The recombinant protein was expressed in high levels in mammalian HEK 293T cells, where it exhibited the pharmacological properties of the native transporter. EAAT2 was purified from isolated cell membranes in a single step using nickel affinity chromatography. In-gel and in-solution trypsin digestions were conducted on the isolated protein and then analyzed by MALDI-TOF and LC-MS/MS mass spectrometry. Overall, 89% sequence coverage of the protein was achieved with these methods. In particular, an 88 amino acid tryptic peptide covering the presumed substrate binding domains HP1, TMD7, HP2, and TMD8 domains of EAAT2 was also identified after N-deglycosylation. Beyond the specific applicability to EAAT2, this study provides an efficient, simple and scalable approach to express, purify, digest and characterize integral membrane transporter proteins by mass spectrometry.

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.

Methamphetamine (METH) use, referred to as methamphetamine use disorder (MUD), results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders (HAND). MUD exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for >90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-κB, transcription factors activated by cAMP, calcium and IL-1β. Second messengers, cAMP and calcium, are triggered by TAAR1 activation, which is upregulated by IL-1β METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference and pharmacological inhibitors targeting or blocking cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Overall, this work revealed METH-induced differential CREB phosphorylation is a critical regulator for EAAT-2 function and may thus serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.

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.