Takahashi K , Chen L , Sayama M , Wu M , Hayashi MK , Irie T , Ohwada T , Sato K .

             glutamate binding

	Figure 1. Characterization of the effect of docosahexaenoic acid (DHA) on EAAT2 current. A, a1, representative traces of L-glutamate (L-Glu, 50 μM for 2 min, black bar)-induced current obtained from Xenopus oocytes over-expressing EAAT2 clamped at −50 mV in the absence or presence of DHA (100 μM for 2 min, gray bar). When co-applied, DHA increased the L-Glu-induced EAAT2 current, and the effect disappeared after washout. The structure of DHA is also shown. a2, effect of DHA (100 μM) on the L-Glu concentration–response curves of EAAT2 current at −50 mV. DHA caused a significant decrease of Km for L-Glu, without significantly affecting the mean maximal current, Imax. Km is the Michaelis constant, which is L-Glu concentration needed to achieve a half-maximum binding at equilibrium. a3, representative traces of L-Glu (50 μM for 2 min, black bars)-induced EAAT1 current in the absence or presence of DHA (100 μM for 2 min, gray bars). When the compounds were co-applied, DHA tended to decrease EAAT1 current, and the effect disappeared after washout. Effects of DHA (100 μM) on the L-Glu-induced current amplitudes of EAAT1. The amplitudes were normalized to those just before the application of DHA. B, b1, concentration-dependence of the effect of DHA on L-Glu induced EAAT2 current. b2, current–voltage relationship for L-Glu-induced EAAT2 current in the absence or presence of DHA (100 μM). The effect of DHA was independent of holding potential. b3, in the presence of dihydrokainic acid (DHK, 100 μM), a selective inhibitor of EAAT2, DHA no longer enhanced the L-Glu-induced EAAT2 current. C, c1, comparison of the effects of DHA and that of DHA analog. The conjugate of DHA with coenzyme A (DHA-CoA, 100 μM), membrane-impermeable analog of DHA, increased the EAAT2 current to almost the same extent as DHA. DHA methyl ester (DHA-ME, 200 μM), an uncharged analog of DHA, had no effect on L-Glu-induced EAAT2 current. c2, the effect of DHA disappeared when the pH was changed from 7.5 to 5.5. c3, the structures of DHA and the analogs used in these experiments. Error bars represent mean ± SD. The numbers written within parentheses in each Figure represent the number of independent experiments. Statistical differences between groups were determined by two-tailed paired Student’s t test (a2, b3, c1, c2) (denoted by ∗). p-values are indicated in each Figure panel. EAAT1, excitatory amino acid transporter subtype 1.
	Figure 2. Leu434 residue in re-entrant hairpin loops HP2a is essential for the augmenting effect of DHA on L-Glu-induced EAAT2 current. A, a1, topology of EAAT1, EAAT2, and EAAT1-EAAT2 hybrid chimeras: EAAT2(EAAT1 TM7b-HP2a), EAAT1(EAAT2 TM7b-HP2a), and EAAT1(EAAT2 connector). a2, the effect of DHA on L-Glu-induced currents of EAAT1, EAAT1(EAAT2 TM7b-HP2a), EAAT1(EAAT2 connector), and EAAT2. Data are shown as rates of increase by DHA. B, b1, 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 EAAT1(EAAT2 TM7b-HP2a) chimera. b2, comparison of the effects of DHA on EAAT1(EAAT2 TM7b-HP2a) chimera and a series of EAAT1(EAAT2 TM7b-HP2a)s with point back mutations to the original amino acid of EAAT1 for the six amino acids shown in b1. Only EAAT1(EAAT2 TM7b-HP2a) L434A shows complete loss of the augmenting effect of DHA. Data are shown as rates of increase by DHA. Exact p-values were 0.7809 for V407L, 0.1898 for M415V, 0.1575 for V426I, 0.4775 for V428I, 0.3042 for L430I, and 0.0006 for L434A. versus EAAT1(EAAT2 TM7b-HP2a group). b3, (top) topology of EAAT2 L434A; (bottom) comparison of the effects of DHA on EAAT2 and EAAT2 L434A. Data are shown as rates of increase by DHA. b4, (top) topology of EAAT1 A435L; (bottom) comparison of the effects of DHA on EAAT1 and EAAT1 A435L. Data are shown as rates of increase by DHA. Error bars represent mean ± SD. The numbers written within parentheses in each Figure represent the number of independent experiments. Statistical differences between groups were determined by two-tailed unpaired Student’s t test (b3 and b4), and Tukey’s test following one-way factorial ANOVA (a2 and b2) (denoted by ∗). p-values are indicated in each Figure panel. DHA, docosahexaenoic acid; EAAT1, excitatory amino acid transporter subtype 1; EAAT2, excitatory amino acid transporter subtype 2.
	Figure 3. Identification of other PUFAs that augment L-Glu-induced EAAT2 current. A, structures of fatty acids used in this experiment. ALA, α-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid, DTetA, docosatetraenoic acid, DTriA, docosatrienoic acid, EPA, eicosapentaenoic acid; OLE, oleic acid. B, b1, effects of fatty acids (100 μM) shown in A on the L-Glu-induced EAAT2 current. DHA, DPA, EPA, ARA, and ALA significantly increased the current. b2, loss of the augmenting effects of DPA, EPA, ARA and ALA in EAAT2 L434A. C, c1, effects of DHA, DPA, EPA, ARA, and ALA (100 μM) on the L-Glu-induced EAAT1 current. DHA significantly decreased the current, while DPA, EPA, ARA, and ALA tended to decrease the current. c2, the effects of DPA, EPA, ARA, and ALA on L-Glu-induced EAAT1 A435L current. These PUFAs augmented the L-Glu-induced EAAT1 A435L current. Error bars represent mean ± SD. The numbers written within parentheses in each Figure represent the number of independent experiments. Statistical differences between groups were determined by two-tailed unpaired Student’s t test (b2 and c2), and Tukey’s test following one-way factorial ANOVA (b1 and c1 versus OLE-treated group) (denoted by ∗). p-values are indicated in each Figure panel. EAAT2, excitatory amino acid transporter subtype 2; PUFA, polyunsaturated fatty acid.
	Figure 4. Proposed binding conformation for DHA in the transport/trimerization domain interface of EAAT2 homology model in the outward facing state OFS. A, a1, extracellular view of trimerized EAAT2 OFS homology model based on EAAT1 crystal structure. Trimerization domain is shown in green ribbon. Transporter domain is shown in gray surface. Structural data were presented using graphical user interface in Maestro Suite. The homology model of EAAT2 was constructed as a monomer based on the crystal structure of OFS EAAT1 (PDBID: 5LLM) with energy-based loop refinement using Homology Modeling unit in Maestro Suite. The quality of homology model was checked by PROCHECK. a2 and a3, magnified monomer in the hatched square in a1 in the absence (a2) or presence (a3) of DHA. The lipid crevice calculated by SiteMap exists at the interface between trimerization domains and transport domains (yellow space) (a2). DHA is docked to the lipid crevice (carbon: purple spheres; hydrogen: white spheres) (a3). B, b1 and b2, docking poses of DHA to the lipid pocket in the vicinity of HP2 according to induced fit docking protocol. The trimerization domain and transport domain are shown in green and gray ribbons, respectively. 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: hiding. Na+ is shown as a pink sphere. Two types of the DHA conformations could be visualized according to the position of the carboxylic group, i.e., one is with carboxyl group on upper side (b1) and the other is with carboxyl group on lower side (b2). Both of them have similar U-shaped conformation. Inset is the DHA conformations in each case. Three-dimensional position of DHA is in close proximity to L-Glu binding site and Na+ binding site. DHA, docosahexaenoic acid; EAAT2, excitatory amino acid transporter subtype 2; OFS, outward facing state.
	Figure 1. Characterization of the effect of docosahexaenoic acid (DHA) on EAAT2 current.A, a1, representative traces of L-glutamate (L-Glu, 50 μM for 2 min, black bar)-induced current obtained from Xenopus oocytes over-expressing EAAT2 clamped at −50 mV in the absence or presence of DHA (100 μM for 2 min, gray bar). When co-applied, DHA increased the L-Glu-induced EAAT2 current, and the effect disappeared after washout. The structure of DHA is also shown. a2, effect of DHA (100 μM) on the L-Glu concentration–response curves of EAAT2 current at −50 mV. DHA caused a significant decrease of Km for L-Glu, without significantly affecting the mean maximal current, Imax. Km is the Michaelis constant, which is L-Glu concentration needed to achieve a half-maximum binding at equilibrium. a3, representative traces of L-Glu (50 μM for 2 min, black bars)-induced EAAT1 current in the absence or presence of DHA (100 μM for 2 min, gray bars). When the compounds were co-applied, DHA tended to decrease EAAT1 current, and the effect disappeared after washout. Effects of DHA (100 μM) on the L-Glu-induced current amplitudes of EAAT1. The amplitudes were normalized to those just before the application of DHA. B, b1, concentration-dependence of the effect of DHA on L-Glu induced EAAT2 current. b2, current–voltage relationship for L-Glu-induced EAAT2 current in the absence or presence of DHA (100 μM). The effect of DHA was independent of holding potential. b3, in the presence of dihydrokainic acid (DHK, 100 μM), a selective inhibitor of EAAT2, DHA no longer enhanced the L-Glu-induced EAAT2 current. C, c1, comparison of the effects of DHA and that of DHA analog. The conjugate of DHA with coenzyme A (DHA-CoA, 100 μM), membrane-impermeable analog of DHA, increased the EAAT2 current to almost the same extent as DHA. DHA methyl ester (DHA-ME, 200 μM), an uncharged analog of DHA, had no effect on L-Glu-induced EAAT2 current. c2, the effect of DHA disappeared when the pH was changed from 7.5 to 5.5. c3, the structures of DHA and the analogs used in these experiments. Error bars represent mean ± SD. The numbers written within parentheses in each Figure represent the number of independent experiments. Statistical differences between groups were determined by two-tailed paired Student’s t test (a2, b3, c1, c2) (denoted by ∗). p-values are indicated in each Figure panel. EAAT1, excitatory amino acid transporter subtype 1.
	Figure 2. Leu434 residue in re-entrant hairpin loops HP2a is essential for the augmenting effect of DHA on L-Glu-induced EAAT2 current.A, a1, topology of EAAT1, EAAT2, and EAAT1-EAAT2 hybrid chimeras: EAAT2(EAAT1 TM7b-HP2a), EAAT1(EAAT2 TM7b-HP2a), and EAAT1(EAAT2 connector). a2, the effect of DHA on L-Glu-induced currents of EAAT1, EAAT1(EAAT2 TM7b-HP2a), EAAT1(EAAT2 connector), and EAAT2. Data are shown as rates of increase by DHA. B, b1, 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 EAAT1(EAAT2 TM7b-HP2a) chimera. b2, comparison of the effects of DHA on EAAT1(EAAT2 TM7b-HP2a) chimera and a series of EAAT1(EAAT2 TM7b-HP2a)s with point back mutations to the original amino acid of EAAT1 for the six amino acids shown in b1. Only EAAT1(EAAT2 TM7b-HP2a) L434A shows complete loss of the augmenting effect of DHA. Data are shown as rates of increase by DHA. Exact p-values were 0.7809 for V407L, 0.1898 for M415V, 0.1575 for V426I, 0.4775 for V428I, 0.3042 for L430I, and 0.0006 for L434A. versus EAAT1(EAAT2 TM7b-HP2a group). b3, (top) topology of EAAT2 L434A; (bottom) comparison of the effects of DHA on EAAT2 and EAAT2 L434A. Data are shown as rates of increase by DHA. b4, (top) topology of EAAT1 A435L; (bottom) comparison of the effects of DHA on EAAT1 and EAAT1 A435L. Data are shown as rates of increase by DHA. Error bars represent mean ± SD. The numbers written within parentheses in each Figure represent the number of independent experiments. Statistical differences between groups were determined by two-tailed unpaired Student’s t test (b3 and b4), and Tukey’s test following one-way factorial ANOVA (a2 and b2) (denoted by ∗). p-values are indicated in each Figure panel. DHA, docosahexaenoic acid; EAAT1, excitatory amino acid transporter subtype 1; EAAT2, excitatory amino acid transporter subtype 2.
	Figure 3. Identification of other PUFAs that augment L-Glu-induced EAAT2 current.A, structures of fatty acids used in this experiment. ALA, α-linolenic acid; ARA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid, DTetA, docosatetraenoic acid, DTriA, docosatrienoic acid, EPA, eicosapentaenoic acid; OLE, oleic acid. B, b1, effects of fatty acids (100 μM) shown in A on the L-Glu-induced EAAT2 current. DHA, DPA, EPA, ARA, and ALA significantly increased the current. b2, loss of the augmenting effects of DPA, EPA, ARA and ALA in EAAT2 L434A. C, c1, effects of DHA, DPA, EPA, ARA, and ALA (100 μM) on the L-Glu-induced EAAT1 current. DHA significantly decreased the current, while DPA, EPA, ARA, and ALA tended to decrease the current. c2, the effects of DPA, EPA, ARA, and ALA on L-Glu-induced EAAT1 A435L current. These PUFAs augmented the L-Glu-induced EAAT1 A435L current. Error bars represent mean ± SD. The numbers written within parentheses in each Figure represent the number of independent experiments. Statistical differences between groups were determined by two-tailed unpaired Student’s t test (b2 and c2), and Tukey’s test following one-way factorial ANOVA (b1 and c1 versus OLE-treated group) (denoted by ∗). p-values are indicated in each Figure panel. EAAT2, excitatory amino acid transporter subtype 2; PUFA, polyunsaturated fatty acid.
	Figure 4. Proposed binding conformation for DHA in the transport/trimerization domain interface of EAAT2 homology model in the outward facing state OFS.A, a1, extracellular view of trimerized EAAT2 OFS homology model based on EAAT1 crystal structure. Trimerization domain is shown in green ribbon. Transporter domain is shown in gray surface. Structural data were presented using graphical user interface in Maestro Suite. The homology model of EAAT2 was constructed as a monomer based on the crystal structure of OFS EAAT1 (PDBID: 5LLM) with energy-based loop refinement using Homology Modeling unit in Maestro Suite. The quality of homology model was checked by PROCHECK. a2 and a3, magnified monomer in the hatched square in a1 in the absence (a2) or presence (a3) of DHA. The lipid crevice calculated by SiteMap exists at the interface between trimerization domains and transport domains (yellow space) (a2). DHA is docked to the lipid crevice (carbon: purple spheres; hydrogen: white spheres) (a3). B, b1 and b2, docking poses of DHA to the lipid pocket in the vicinity of HP2 according to induced fit docking protocol. The trimerization domain and transport domain are shown in green and gray ribbons, respectively. 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: hiding. Na+ is shown as a pink sphere. Two types of the DHA conformations could be visualized according to the position of the carboxylic group, i.e., one is with carboxyl group on upper side (b1) and the other is with carboxyl group on lower side (b2). Both of them have similar U-shaped conformation. Inset is the DHA conformations in each case. Three-dimensional position of DHA is in close proximity to L-Glu binding site and Na+ binding site. DHA, docosahexaenoic acid; EAAT2, excitatory amino acid transporter subtype 2; OFS, outward facing state.

Akyuz, Transport domain unlocking sets the uptake rate of an aspartate transporter. 2015, Pubmed

Akyuz, Transport domain unlocking sets the uptake rate of an aspartate transporter. 2015, Pubmed
Akyuz, Transport dynamics in a glutamate transporter homologue. 2013, Pubmed
Arriza, Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. 1997, Pubmed , Xenbase
Arriza, Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. 1994, Pubmed , Xenbase
Barish, A transient calcium-dependent chloride current in the immature Xenopus oocyte. 1983, Pubmed , Xenbase
Bazinet, Polyunsaturated fatty acids and their metabolites in brain function and disease. 2014, Pubmed
Berry, Differential modulation of the glutamate transporters GLT1, GLAST and EAAC1 by docosahexaenoic acid. 2005, Pubmed
Boland, Polyunsaturated fatty acid modulation of voltage-gated ion channels. 2008, Pubmed
Börjesson, Lipoelectric modification of ion channel voltage gating by polyunsaturated fatty acids. 2008, Pubmed , Xenbase
Börjesson, Electrostatic tuning of cellular excitability. 2010, Pubmed , Xenbase
Börjesson, Structure, function, and modification of the voltage sensor in voltage-gated ion channels. 2008, Pubmed
Börjesson, An electrostatic potassium channel opener targeting the final voltage sensor transition. 2011, Pubmed , Xenbase
Boudker, Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. 2007, Pubmed
Canul-Tec, Structure and allosteric inhibition of excitatory amino acid transporter 1. 2017, Pubmed
Cao, Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function. 2009, Pubmed
Danbolt, Glutamate uptake. 2001, Pubmed
Dascal, Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes. 1986, Pubmed , Xenbase
Eldho, Polyunsaturated docosahexaenoic vs docosapentaenoic acid-differences in lipid matrix properties from the loss of one double bond. 2003, Pubmed
Englund, A voltage dependent non-inactivating Na+ channel activated during apoptosis in Xenopus oocytes. 2014, Pubmed , Xenbase
Fairbairn, Combining a high DHA multi-nutrient supplement with aerobic exercise: Protocol for a randomised controlled study assessing mobility and cognitive function in older women. 2019, Pubmed
Fairman, Arachidonic acid elicits a substrate-gated proton current associated with the glutamate transporter EAAT4. 1998, Pubmed , Xenbase
Fairman, An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. 1995, Pubmed , Xenbase
Feller, Polyunsaturated fatty acids in lipid bilayers: intrinsic and environmental contributions to their unique physical properties. 2002, Pubmed
Friesner, Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. 2004, Pubmed
Grewer, Electrogenic glutamate transporters in the CNS: molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling. 2005, Pubmed
Grewer, Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds. 2000, Pubmed
Guizy, Modulation of the atrial specific Kv1.5 channel by the n-3 polyunsaturated fatty acid, alpha-linolenic acid. 2008, Pubmed
Heckman, Gene splicing and mutagenesis by PCR-driven overlap extension. 2007, Pubmed
Holmseth, The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS. 2012, Pubmed
Kanner, Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain. 1982, Pubmed
Kawanabe, Effects of unsaturated fatty acids on the kinetics of voltage-gated proton channels heterologously expressed in cultured cells. 2016, Pubmed
Kortzak, Allosteric gate modulation confers K+ coupling in glutamate transporters. 2019, Pubmed
Laskowski, AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. 1996, Pubmed
Levy, Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. 1998, Pubmed
Mayol, Cholesterol and omega-3 fatty acids inhibit Na, K-ATPase activity in human endothelial cells. 1999, Pubmed
McKhann, Heterogeneity of astrocyte resting membrane potentials and intercellular coupling revealed by whole-cell and gramicidin-perforated patch recordings from cultured neocortical and hippocampal slice astrocytes. 1997, Pubmed
McNamara, Cognitive response to fish oil, blueberry, and combined supplementation in older adults with subjective cognitive impairment. 2018, Pubmed
Miledi, A calcium-dependent transient outward current in Xenopus laevis oocytes. 1982, Pubmed , Xenbase
Mitrovic, Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2. 1998, Pubmed , Xenbase
Reyes, Transport mechanism of a bacterial homologue of glutamate transporters. 2009, Pubmed
Robinson, Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain. 1991, Pubmed
Rong, Investigating substrate-induced motion between the scaffold and transport domains in the glutamate transporter EAAT1. 2014, Pubmed
Sherman, Novel procedure for modeling ligand/receptor induced fit effects. 2006, Pubmed
Sherman, Use of an induced fit receptor structure in virtual screening. 2006, Pubmed
Tanaka, Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. 1997, Pubmed
Tanui, Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1. 2016, Pubmed
Tzingounis, Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4. 1998, Pubmed , Xenbase
Vandenberg, Mechanisms of glutamate transport. 2013, Pubmed
Verdon, Crystal structure of an asymmetric trimer of a bacterial glutamate transporter homolog. 2012, Pubmed
Wadiche, Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel. 1998, Pubmed , Xenbase
Wadiche, Ion fluxes associated with excitatory amino acid transport. 1995, Pubmed , Xenbase
Wang, Energy Landscape of the Substrate Translocation Equilibrium of Plasma-Membrane Glutamate Transporters. 2018, Pubmed
Wurtman, Synapse formation in the brain can be enhanced by co-administering three specific nutrients. 2017, Pubmed
Yazdi, The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel. 2016, Pubmed
Yernool, Structure of a glutamate transporter homologue from Pyrococcus horikoshii. 2004, Pubmed
Yurko-Mauro, Docosahexaenoic acid and adult memory: a systematic review and meta-analysis. 2015, Pubmed
Zerangue, Differential modulation of human glutamate transporter subtypes by arachidonic acid. 1995, Pubmed , Xenbase
Zerangue, Flux coupling in a neuronal glutamate transporter. 1996, Pubmed , Xenbase