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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. EAAT2 is an amino acid transporter implicated in glutamate homeostasis in brain and therapy resistance of cancer cells. Here, the authors report cryo-EM structures and reveal inhibitory mechanisms via selective inhibitor WAY213613.

The excitatory amino acid transporter 2 (EAAT2) is the major glutamate transporter in the brain expressed predominantly in astrocytes and at low levels in neurons and axonal terminals. EAAT2 expression is reduced in aging and sporadic Alzheimer’s disease (AD) patients’ brains. The role EAAT2 plays in cognitive aging and its associated mechanisms remains largely unknown. Here, we show that conditional deletion of astrocytic and neuronal EAAT2 results in age-related cognitive deficits. Astrocytic, but not neuronal EAAT2, deletion leads to early deficits in short-term memory and in spatial reference learning and long-term memory. Neuronal EAAT2 loss results in late-onset spatial reference long-term memory deficit. Neuronal EAAT2 deletion leads to dysregulation of the kynurenine pathway, and astrocytic EAAT2 deficiency results in dysfunction of innate and adaptive immune pathways, which correlate with cognitive decline. Astrocytic EAAT2 deficiency also shows transcriptomic overlaps with human aging and AD. Overall, the present study shows that in addition to the widely recognized astrocytic EAAT2, neuronal EAAT2 plays a role in hippocampus-dependent memory. Furthermore, the gene expression profiles associated with astrocytic and neuronal EAAT2 deletion are substantially different, with the former associated with inflammation and synaptic function similar to changes observed in human AD and gene expression changes associated with inflammation similar to the aging human brain.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt Ph and Glt Tk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST(CreERT2/+)/GLT1(flox/flox), iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.

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. High-resolution structures of the thermostabilized human excitatory amino acid transporter EAAT1, alone or in association with its substrate or small molecule inhibitors, reveal architectural features of human SLC1 transporters and an allosteric mechanism of inhibition. Structure of an amino acid transporter Amino acid transporters of the solute carrier 1 (SLC1) family have been associated with several neurological and metabolic disorders in humans, but information about their structure has been limited to a simpler homologue from an archeal microorganism. Nicolas Reyes and colleagues present several high-resolution structures of the human excitatory amino acid transporter 1 (EAAT1), a key component of glutamatergic synapses, alone or in association with its substrate or small inhibitor molecules. The structures reveal mechanistic determinants that are specific to human SLC1 carriers, such as regulation by lipids or post-translational modifications, and present an allosteric pocket that could aid further drug design. On the basis of these structures, researchers will be able to propose how specific mutations affect EAAT1 transport mechanics at a molecular level and therefore suggest more effective treatment approaches.

ObjectiveGlutamate transporters play a crucial role in neurotransmitter homeostasis, but studying their structure and function is challenging due to their membrane-bound nature. This study aims to investigate whether water-soluble QTY-variants of glutamate transporters EAA1, EAA2 and EAA3 retain the conformational characteristics and dynamics of native membrane-bound transporters.MethodsMolecular dynamics simulations and comparative genomics were used to analyze the structural dynamics of both native transporters and their QTY-variants. Native transporters were simulated in lipid bilayers, while QTY-variants were simulated in aqueous solution. Lipid distortions, relative solvent accessibilities, and conformational changes were examined. Evolutionary conservation profiles were correlated with structural dynamics. Statistical analyses included multivariate analysis to account for confounding variables.ResultsQTY-variants exhibited similar residue-wise conformational dynamics to their native counterparts, with correlation coefficients of 0.73 and 0.56 for EAA1 and EAA3, respectively (p < 0.001). Hydrophobic interactions of native helices correlated with water interactions of QTY- helices (rs = 0.4753, p < 0.001 for EAA1). QTY-variants underwent conformational changes resembling the outward-to-inward transition of native transporters.ConclusionsWater-soluble QTY-variants retain key structural properties of native glutamate transporters and mimic aspects of native lipid interactions, including conformational flexibility. This research provides valuable insights into the conformational changes and molecular mechanisms of glutamate transport, potentially offering a new approach for studying membrane protein dynamics and drug interactions.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity 1 . The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism 2 – 5 . Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport 6 – 8 . However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family. Glutamate transporters conduct chloride ions through an aqueous channel with hydrophobic gates that forms during the glutamate transport cycle.

Cardiac arrest (CA)-induced post-cardiac arrest brain injury (PCABI) represents a critical contributor to global mortality and neurological disability. While sleep deprivation (SD) is recognized to aggravate neurological outcomes, its role in PCABI pathogenesis remains underexplored. This study investigated the mechanisms by which SD exacerbates PCABI and evaluated the neuroprotective efficacy of electroacupuncture (EA). A CA model was established in SD rats, followed by RNA sequencing and molecular analyses to assess brain injury biomarkers, synaptic plasticity, and calcium signaling pathways. SD disrupted circadian rhythms, amplified neuronal apoptosis, and suppressed glutamate transporter Excitatory Amino Acid Transporter 2 (EAAT2) expression post-CA, correlating with worsened cognitive deficits. EA treatment significantly attenuated these effects, restoring EAAT2 levels, mitigating calcium overload, and enhancing synaptic integrity. Mechanistically, EA modulated the EAAT2/calcium signaling axis and rebalanced autonomic nervous activity, thereby reducing oxidative stress and neuronal excitotoxicity. These findings identify EAAT2 downregulation as a key mediator of SD-aggravated PCABI and establish EA as a dual-target intervention that rectifies glutamatergic dysregulation and autonomic dysfunction. The study provides translational insights into EA’s therapeutic potential for PCABI, particularly in populations with comorbid sleep disturbances.

The excessive glutamate-mediated excitotoxicity is a major cause of the neuron death in ischemic stroke (IS). Astrocytic glutamate transporter protein-1 (GLT-1, also named excitatory amino acid transporter 2, EAAT2) is essential for maintaining low extracellular glutamate and preventing glutamate neurotoxicity, while its expression is regulated by Yes-associated protein (YAP) signaling reported by our previous study. Recent studies have shown that ischemic injury of the brain induces the gain of stemness in astrocytes dependent on the de novo DNA methyltransferase DNMT3A, and YAP signaling contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation. However, it remains unknown the roles of astrocytic YAP signaling in IS and whether it regulates the glutamate-mediated excitotoxicity and the gain of stemness in astrocytes induced by IS. In this study, we found that IS was aggravated in YAP GFAP -CKO mice with inhibition of the functional behavioral recovery, larger injury area, more apoptotic neurons and more inflammatory infiltration. Furthermore, YAP deletion in astrocytes impaired the formation of glial scars due to the reduction of astrocytic proliferation, and inhibition of activation and gain of stemness in astrocytes induced by IS. Additionally, the expression of EAAT2 was significantly decreased in the cortical astrocytes of YAP GFAP -CKO mice after IS through downregulating β-catenin signaling. Activation of EAAT2 by LDN-212320 partially restored the deficits such as neuronal death and behavioral recovery impairment in YAP GFAP -CKO mice after IS. Furthermore, activation of astrocytic YAP signaling by XMU-MP-1 upregulated the EAAT2 expression, and inhibited the loss of neurons, and promoted the gain of stemness in astrocytes and functional recovery of mice in IS. These results identify that astrocytic YAP signaling prevents the glutamate neurotoxicity by upregulating EAAT2 expression and promotes the gain of stemness in astrocytes in IS, which provides a novel drug target for IS treatment.

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). Excitatory amino acid transporters (EAATs) have important roles in the uptake of glutamate and termination of glutamatergic transmission. Up to now, five EAAT isoforms (EAAT1-5) have been identified in mammals. The main focus of this review is EAAT2. This protein has an important role in the pathoetiology of epilepsy. De novo dominant mutations, as well as inherited recessive mutation in this gene, have been associated with epilepsy. Moreover, dysregulation of this protein is implicated in a range of neurological diseases, namely amyotrophic lateral sclerosis, alzheimer’s disease, parkinson’s disease, schizophrenia, epilepsy, and autism. In this review, we summarize the role of EAAT2 in epilepsy and other neurological disorders, then provide an overview of the therapeutic modulation of this protein.