Takahashi K , Sato K .

Figure 1. (A) (a1). Oocytes were collected from anaesthetized Xenopus laevis. The isolated oocytes were then treated with collagenase (2 mg mL−1, type 1), and capped mRNA was injected into either defolliculated stage V or VI oocytes. The oocytes were incubated for 2–7 d at 18 °C in ND96 solution containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.5) supplemented with 0.01% gentamycin. TEVC recordings from the oocytes were performed at room temperature (25 °C) using glass microelectrodes filled with 3 M KCl (resistance = 1–4 MΩ) and an Ag/AgCl pellet electrode. (a2). Substrate- and the coupling ion-transports by EAATs. Substrate, such as L-Glu, L-Asp, or D-Asp, transport through EAATs is coupled to the co-transport of 3 Na+ and 1 H+ followed by the counter transport of 1 K+. In addition, the binding of substrates and Na+ to EAATs activates uncoupled Cl− anion currents. (B) (b1). A representative trace of L-Glu (50 μM for 2 min, black bar)-induced current obtained from Xenopus oocytes overexpressing EAAT2 clamped at −50 mV. (b2). IV relationship for L-Glu (50 μM)-induced EAAT2 current. To examine the IV relationship, the L-Glu-induced current was calculated through the subtraction of the steady-state current from the L-Glu-induced current. The curves were obtained with a holding potential of −60 mV applying an 8000 ms ramp pulse from −110 to +60 mV. Data are shown as the values normalized to that obtained with 50 μM L-Glu at −100 mV. Means, n = 5. (C) (c1). Model curves of transport-induced currents. The total L-Glu-induced EAAT currents (solid line: Itotal), electrophysiologically recorded using TEVC methods from EAAT-expressing Xenopus oocytes, represent the sum of the coupled L-Glu transport currents (dotted line: IAA) and the uncoupled Cl− anion currents (dotted line: ICl). (c2). The predicted reversal potential of the net current (Itotal) is independent of substrate concentration when the concentration dependence of IAA and ICl is the same. However, the amplitude of Itotal is dependent on the substrate concentration. (c3). The absolute reversal potential of Itotal is dependent on IAA relative to ICl [14] (copyright permission has been obtained). (D) (d1). D-Asp uptake and charge translocation were simultaneously measured during a 100 s application of 100 μM [3H] D-Asp to voltage-clamped oocytes expressing EAAT1 under 104 mM Cl− conditions. (d2). Voltage dependence of RI-labelled D-Asp flux and superimposed exponential (e-fold/75 mV) derived from fit of transport current under nominal Cl−-free conditions [14] (copyright permission has been obtained). (E) Overall structure of human EAAT2 as viewed from the membrane plane (left) and the intracellular side (right). The trimerization domain is blue and the transport domain is red. The protomer of EAAT2 is divided into two distinct functional components: one is a rigid scaffold domain that mediates interprotomer interactions and is located in the center of the trimer, and the other is a transport domain containing the substrate-binding site [15]. (F) Schematic representation of the transport cycle of EAATs (elevator motion). The transport domain (red) moves across the membrane relative to the trimerization domain (blue). The transported L-Glu is pink. When L-Glu binds to the transport domain of the EAAT in outward facing state (top), the conformation changes to occluded state (middle) first, then changes to inward facing state (bottom), thereby releasing L-Glu into the cell.

Figure 2. (A) The chemical structures of DHA and DHA-CoA. (B) Representative traces of L-glutamate (L-Glu, 50 μM for 2 min, black horizontal line)-induced current obtained from Xenopus oocytes overexpressing EAAT2 clamped at −50 mV in the absence or presence of DHA (100 μM for 2 min, grey horizontal line). When coapplied, DHA increased the L-Glu-induced EAAT2 current, and the effect disappeared after washout. (C) Representative traces of L-Glu-induced EAAT1 currents in the absence or presence of DHA. When the compounds were coapplied, DHA tended to decrease the EAAT1 current, and the effect disappeared after washout. (D) Topology of EAAT1, EAAT2 (d1). EAAT1/2 is organized into eight transmembrane (TM1-8) and two helical hairpins (HP1 and HP2), which are re-entrant loops. TM7b-HP2a sequence and connector sequence in the red square. d2-d4. EAAT1-EAAT2 hybrid chimeras: EAAT2 (EAAT1 TM7b-HP2a) (d2), EAAT1 (EAAT2 TM7b-HP2a) (d3), and EAAT1 (EAAT2 connector) (d4). (E) Amino acid alignment from TM7b to HP2a of EAAT2 and EAAT1. The common amino acids are shown on a black background. Single amino acid back-mutations were performed at the sites indicated by black arrowheads in the EAAT1 (EAAT2 TM7b-HP2a) chimaera. (F) Left, topology of EAAT2 L434A. Right, comparison of the effects of DHA on EAAT2 and EAAT2 L434A. Data are shown as rates of increase by DHA. (G) Left, topology of EAAT1 A435L. Right, comparison of the effects of DHA on EAAT1 and EAAT1 A435L. Data are shown as rates of increase by DHA. (H) Proposed binding conformation for DHA in the transport domain/scaffold domain interface of the EAAT2 homology model in the outward facing state (OFS). (h1). Extracellular view of the trimerized EAAT2 OFS homology model based on the EAAT1 crystal structure. The scaffold domain is shown as a green ribbon. The transport domain is shown as a grey surface. (h2). Magnified monomer in the hatched square in h1 in the presence of DHA. The lipid crevice calculated by SiteMap exists at the interface between the scaffold domain and the transport domain (yellow space). DHA is docked to the lipid crevice (carbon: purple spheres; hydrogen: white spheres). (I) Docking poses of DHA in the lipid pocket in the vicinity of HP2 according to the induced fit docking protocol. The scaffold domain and transport domain are shown in green and grey ribbons, respectively. The carbons in DHA and EAAT2 L434 are represented by purple and yellow sticks, respectively. The atoms in L-Glu are shown as follows: carbon, blue sphere; hydrogen, white sphere; oxygen, red sphere; nitrogen, hidden. Na+ is shown as a pink sphere. Two types of DHA conformations could be visualized according to the position of the carboxylic group, i.e., one with a carboxyl group on the upper side (i1) and the other with a carboxyl group on the lower side (i2). Both of them have similar U-shaped conformations. The inset shows the DHA conformations in each case. The three-dimensional position of DHA is in close proximity to the L-Glu binding site and Na+ binding site. Error bars represent the mean ±SD. The numbers written within parentheses in the respective figures represent the number of independent experiments. Statistical differences between groups were determined by two-tailed paired Student’s t test. p values are indicated in each figure panel [58].

Abrahamsen, Allosteric modulation of an excitatory amino acid transporter: the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domain. 2013, Pubmed

Abrahamsen, Allosteric modulation of an excitatory amino acid transporter: the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domain. 2013, Pubmed
Akabas, Cysteine Modification: Probing Channel Structure, Function and Conformational Change. 2015, Pubmed
Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed , Xenbase
Akyuz, Transport domain unlocking sets the uptake rate of an aspartate transporter. 2015, Pubmed
Arriza, Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. 1994, Pubmed , Xenbase
Arriza, Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. 1997, Pubmed , Xenbase
Aïd, Effect of a diet-induced n-3 PUFA depletion on cholinergic parameters in the rat hippocampus. 2003, Pubmed
Bazan, Synaptic lipid signaling: significance of polyunsaturated fatty acids and platelet-activating factor. 2003, Pubmed
Berry, Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid. 2005, Pubmed
Boudker, Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. 2007, Pubmed
Bridges, The excitatory amino acid transporters: pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes. 2005, Pubmed
Bridges, A pharmacological review of competitive inhibitors and substrates of high-affinity, sodium-dependent glutamate transport in the central nervous system. 1999, Pubmed
Brocke, Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis. 2002, Pubmed
Bunch, Excitatory amino acid transporters as potential drug targets. 2009, Pubmed
Canul-Tec, Structure and allosteric inhibition of excitatory amino acid transporter 1. 2017, Pubmed
Chen, Glutamate transporters have a chloride channel with two hydrophobic gates. 2021, Pubmed , Xenbase
Conti, Shaping excitation at glutamatergic synapses. 1999, Pubmed
Cudkowicz, Safety and efficacy of ceftriaxone for amyotrophic lateral sclerosis: a multi-stage, randomised, double-blind, placebo-controlled trial. 2014, Pubmed
Donevan, The methylglutamate, SYM 2081, is a potent and highly selective agonist at kainate receptors. 1998, Pubmed , Xenbase
Duffield, Transport rate of EAAT2 is regulated by amino acid located at the interface between the scaffolding and substrate transport domains. 2020, Pubmed , Xenbase
Dunlop, Characterization of novel aryl-ether, biaryl, and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high-affinity glutamate transporter EAAT2. 2005, Pubmed , Xenbase
Dunlop, Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter. 1999, Pubmed
Dvorak, An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters. 2021, Pubmed
Fairman, An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. 1995, Pubmed , Xenbase
Fairman, Arachidonic acid elicits a substrate-gated proton current associated with the glutamate transporter EAAT4. 1998, Pubmed , Xenbase
Fontana, Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom. 2007, Pubmed
Fontana, Purification of a neuroprotective component of Parawixia bistriata spider venom that enhances glutamate uptake. 2003, Pubmed
Friesner, Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. 2004, Pubmed
Fujita, Docosahexaenoic acid improves long-term potentiation attenuated by phospholipase A(2) inhibitor in rat hippocampal slices. 2001, Pubmed
Garcia, Huntington's Disease Patient-Derived Astrocytes Display Electrophysiological Impairments and Reduced Neuronal Support. 2019, Pubmed
Gegelashvili, Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. 1997, Pubmed
Green, The emerging role of group VI calcium-independent phospholipase A2 in releasing docosahexaenoic acid from brain phospholipids. 2008, Pubmed
Grunewald, Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology. 1998, Pubmed
Grunewald, Cysteine-scanning mutagenesis reveals a conformationally sensitive reentrant pore-loop in the glutamate transporter GLT-1. 2002, Pubmed
Haugeto, Brain glutamate transporter proteins form homomultimers. 1996, Pubmed
Heider, Human iPSC-Derived Glia as a Tool for Neuropsychiatric Research and Drug Development. 2021, Pubmed
Herman, Extracellular glutamate concentration in hippocampal slice. 2007, Pubmed
Hinoi, Glutamate transporters as drug targets. 2005, Pubmed
Héja, Astrocytes convert network excitation to tonic inhibition of neurons. 2012, Pubmed
Jabaudon, Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin. 1999, Pubmed
Jardetzky, Simple allosteric model for membrane pumps. 1966, Pubmed
Kanai, Primary structure and functional characterization of a high-affinity glutamate transporter. 1992, Pubmed , Xenbase
Kato, Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2. 2022, Pubmed
Koch, Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters. 1999, Pubmed , Xenbase
Kolen, An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates. 2020, Pubmed
Kortagere, Identification of Novel Allosteric Modulators of Glutamate Transporter EAAT2. 2018, Pubmed
Kuratko, The relationship of docosahexaenoic acid (DHA) with learning and behavior in healthy children: a review. 2013, Pubmed
Lehre, Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. 1995, Pubmed
Lin, SLC transporters as therapeutic targets: emerging opportunities. 2015, Pubmed
Magistretti, A cellular perspective on brain energy metabolism and functional imaging. 2015, Pubmed
Mazzocchi-Jones, Impaired corticostriatal LTP and depotentiation following iPLA2 inhibition is restored following acute application of DHA. 2015, Pubmed
Mennerick, Neuronal expression of the glutamate transporter GLT-1 in hippocampal microcultures. 1998, Pubmed
Michaelis, Molecular biology of glutamate receptors in the central nervous system and their role in excitotoxicity, oxidative stress and aging. 1998, Pubmed
Mitrovic, Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2. 1998, Pubmed , Xenbase
Moore, Cerebral endothelium and astrocytes cooperate in supplying docosahexaenoic acid to neurons. 1993, Pubmed
Moore, Polyunsaturated fatty acid synthesis and release by brain-derived cells in vitro. 2001, Pubmed
Novak, High dietary omega-6 fatty acids contribute to reduced docosahexaenoic acid in the developing brain and inhibit secondary neurite growth. 2008, Pubmed
Otis, Isolation of current components and partial reaction cycles in the glial glutamate transporter EAAT2. 2000, Pubmed
Piomelli, Arachidonic acid in cell signaling. 1993, Pubmed
Piomelli, Lipoxygenase metabolites of arachidonic acid in neuronal transmembrane signalling. 1990, Pubmed
Rapoport, Translational studies on regulation of brain docosahexaenoic acid (DHA) metabolism in vivo. 2013, Pubmed
Reyes, Transport mechanism of a bacterial homologue of glutamate transporters. 2009, Pubmed
Rong, TM4 of the glutamate transporter GLT-1 experiences substrate-induced motion during the transport cycle. 2016, Pubmed
Rose, Glutamate transporter coupling to Na,K-ATPase. 2009, Pubmed
Rose, Astroglial Glutamate Signaling and Uptake in the Hippocampus. 2017, Pubmed
Rothstein, Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. 1995, Pubmed
Rothstein, Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. 2005, Pubmed
Salem, Mechanisms of action of docosahexaenoic acid in the nervous system. 2001, Pubmed
Sato, Estrogens inhibit l-glutamate uptake activity of astrocytes via membrane estrogen receptor alpha. 2003, Pubmed
Schlag, Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. 1998, Pubmed
Sherman, Use of an induced fit receptor structure in virtual screening. 2006, Pubmed
Sherman, Novel procedure for modeling ligand/receptor induced fit effects. 2006, Pubmed
Shigeri, Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5). 2001, Pubmed , Xenbase
Shimamoto, DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. 1998, Pubmed , Xenbase
Shimamoto, Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. 2004, Pubmed
Sogaard, Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1. 2013, Pubmed , Xenbase
Storck, Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. 1992, Pubmed , Xenbase
Strokin, Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A2 and is differently regulated by cyclic AMP and Ca2+. 2003, Pubmed
Strokin, Prostaglandin synthesis in rat brain astrocytes is under the control of the n-3 docosahexaenoic acid, released by group VIB calcium-independent phospholipase A2. 2007, Pubmed
Swanson, Neuronal regulation of glutamate transporter subtype expression in astrocytes. 1997, Pubmed
Takahashi, Leucine 434 is essential for docosahexaenoic acid-induced augmentation of L-glutamate transporter current. 2023, Pubmed , Xenbase
Takaki, L-glutamate released from activated microglia downregulates astrocytic L-glutamate transporter expression in neuroinflammation: the 'collusion' hypothesis for increased extracellular L-glutamate concentration in neuroinflammation. 2012, Pubmed
Tanaka, Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. 1997, Pubmed
Trotti, Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? 1998, Pubmed
Trotti, SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivate a glial glutamate transporter. 1999, Pubmed , Xenbase
Tyzack, Human Stem Cell-Derived Astrocytes: Specification and Relevance for Neurological Disorders. 2016, Pubmed
Tzingounis, Glutamate transporters: confining runaway excitation by shaping synaptic transmission. 2007, Pubmed
Valtcheva, Control of Long-Term Plasticity by Glutamate Transporters. 2019, Pubmed
Vandenberg, Mechanisms of glutamate transport. 2013, Pubmed
Vandenberg, Contrasting modes of action of methylglutamate derivatives on the excitatory amino acid transporters, EAAT1 and EAAT2. 1997, Pubmed , Xenbase
Vandenberg, Constitutive ion fluxes and substrate binding domains of human glutamate transporters. 1995, Pubmed , Xenbase
Verdon, Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog. 2012, Pubmed
Voutsinos-Porche, Glial glutamate transporters mediate a functional metabolic crosstalk between neurons and astrocytes in the mouse developing cortex. 2003, Pubmed
Wadiche, Ion fluxes associated with excitatory amino acid transport. 1995, Pubmed , Xenbase
Wadiche, Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel. 1998, Pubmed , Xenbase
Wang, The Druggability of Solute Carriers. 2020, Pubmed
Williard, Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes. 2001, Pubmed
Willis, L-trans-2,3-pyrrolidine dicarboxylate: characterization of a novel excitotoxin. 1996, Pubmed
Wilton, The contribution of glial cells to Huntington's disease pathogenesis. 2020, Pubmed
Yang, Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1. 2009, Pubmed
Yernool, Structure of a glutamate transporter homologue from Pyrococcus horikoshii. 2004, Pubmed
Zerangue, Flux coupling in a neuronal glutamate transporter. 1996, Pubmed , Xenbase
Zhang, Two serine residues of the glutamate transporter GLT-1 are crucial for coupling the fluxes of sodium and the neurotransmitter. 1999, Pubmed
Zhang, Structural basis of ligand binding modes of human EAAT2. 2022, Pubmed