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• W2046055422 abstract "In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na+ ions, followed by countertransport of K+. Recent studies, based on several crystal structures of the archeal homologue GltPh, indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na+ binding, but functional and computational studies suggest some candidate sites. In the GltPh structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of GltPh. Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na+ and K+, respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters. In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na+ ions, followed by countertransport of K+. Recent studies, based on several crystal structures of the archeal homologue GltPh, indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na+ binding, but functional and computational studies suggest some candidate sites. In the GltPh structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of GltPh. Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na+ and K+, respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters. Glutamate is the major excitatory neurotransmitter in the brain. The synaptic actions of this neurotransmitter are terminated by glutamate transporters, which move the transmitter away from the synapse and back into the cells surrounding the synapse and keep its synaptic concentrations below neurotoxic levels. Glutamate transport is an electrogenic process, (1Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar, 2Brew H. Attwell D. Nature. 1987; 327: 707-709Crossref PubMed Scopus (328) Google Scholar, 3Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (350) Google Scholar), which consists of two half-cycles (4Kanner B.I. Bendahan A. Biochemistry. 1982; 21: 6327-6330Crossref PubMed Scopus (166) Google Scholar, 5Pines G. Kanner B.I. Biochemistry. 1990; 29: 11209-11214Crossref PubMed Scopus (91) Google Scholar, 6Kavanaugh M.P. Bendahan A. Zerangue N. Zhang Y. Kanner B.I. J. Biol. Chem. 1997; 272: 1703-1708Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar): (i) cotransport of the neurotransmitter with sodium and hydrogen ions (1Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar, 7Nelson P.J. Dean G.E. Aronson P.S. Rudnick G. Biochemistry. 1983; 22: 5459-5463Crossref PubMed Scopus (50) Google Scholar) and (ii) countertransport of potassium (1Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar, 4Kanner B.I. Bendahan A. Biochemistry. 1982; 21: 6327-6330Crossref PubMed Scopus (166) Google Scholar). The stoichiometry is three sodium ions, one proton, and one potassium ion per transported glutamate molecule (8Zerangue N. Kavanaugh M.P. Nature. 1996; 383: 634-637Crossref PubMed Scopus (708) Google Scholar, 9Levy L.M. Warr O. Attwell D. J. Neurosci. 1998; 18: 9620-9628Crossref PubMed Google Scholar). The sequential translocation mechanism (Fig. 1A) is supported by the fact that mutants impaired in the interaction with potassium are “locked” in an obligatory exchange mode (6Kavanaugh M.P. Bendahan A. Zerangue N. Zhang Y. Kanner B.I. J. Biol. Chem. 1997; 272: 1703-1708Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Zhang Y. Bendahan A. Zarbiv R. Kavanaugh M.P. Kanner B.I. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 751-755Crossref PubMed Scopus (88) Google Scholar). Glutamate transporters mediate two types of substrate-induced steady-state current: an inward-rectifying or “coupled” current, reflecting electrogenic ion-coupled glutamate translocation, and an “uncoupled” sodium-dependent current, which is carried by chloride ions and further activated by the substrates of the transporter (11Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 12Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1014) Google Scholar, 13Arriza J.L. Eliasof S. Kavanaugh M.P. Amara S.G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4155-4160Crossref PubMed Scopus (803) Google Scholar). Nontransportable substrate analogues, expected to “lock” the transporter in an outward-facing conformation when applied from the external side (Fig. 1A, dashed line), are not only competitive inhibitors of the coupled current and of the substrate-induced uncoupled anion current, but also inhibit the basal sodium-dependent anion conductance (14Otis T.S. Kavanaugh M.P. J. Neurosci. 2000; 20: 2749-2757Crossref PubMed Google Scholar, 15Grewer C. Watzke N. Wiessner M. Rauen T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9706-9711Crossref PubMed Scopus (153) Google Scholar). Moreover, such analogues inhibit the sodium-dependent transient currents (3Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (350) Google Scholar), which are thought to reflect a charge-moving conformational change in response to sodium binding. The publication of a high-resolution crystal structure of a glutamate transporter homologue, GltPh, from the archeon Pyrococcus horikoshii represented a landmark for the field of glutamate transporters (16Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar). The structure shows a trimer with a permeation pathway through each of the monomers, indicating that the monomer is the functional unit. This is also the case for the eukaryotic glutamate transporters (17Koch H.P. Larsson H.P. J. Neurosci. 2005; 25: 1730-1736Crossref PubMed Scopus (100) Google Scholar, 18Grewer C. Balani P. Weidenfeller C. Bartusel T. Tao Z. Rauen T. Biochemistry. 2005; 44: 11913-11923Crossref PubMed Scopus (122) Google Scholar, 19Leary G.P. Stone E.F. Holley D.C. Kavanaugh M.P. J. Neurosci. 2007; 27: 2938-2942Crossref PubMed Scopus (74) Google Scholar, 20Koch H.P. Brown R.L. Larsson H.P. J. Neurosci. 2007; 27: 2943-2947Crossref PubMed Scopus (88) Google Scholar). The membrane topology of the monomer (16Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar) is quite unusual but is in excellent agreement with the topology inferred from biochemical studies (21Grunewald M. Bendahan A. Kanner B.I. Neuron. 1998; 21: 623-632Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 22Grunewald M. Kanner B.I. J. Biol. Chem. 2000; 275: 9684-9689Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 23Slotboom D.J. Sobczak I. Konings W.N. Lolkema J.S. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 14282-14287Crossref PubMed Scopus (112) Google Scholar). The monomer contains eight transmembrane domains (TM) 2The abbreviation used is: TMtransmembrane domain. and two oppositely oriented reentrant loops, one between domains 6 and 7 (HP1) and the other between domains 7 and 8 (HP2) (Fig. 1B). TMs 1–6 form the outer shell of the transporter monomer, whereas TMs 7 and 8 and the two reentrant loops participate in the formation of the binding pocket of GltPh (16Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar, 24Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Nature. 2007; 445: 387-393Crossref PubMed Scopus (396) Google Scholar). Importantly, many of the amino acid residues of the transporter, inferred to be important in the interaction with sodium (25Zhang Y. Kanner B.I. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 1710-1715Crossref PubMed Scopus (76) Google Scholar, 26Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), potassium (6Kavanaugh M.P. Bendahan A. Zerangue N. Zhang Y. Kanner B.I. J. Biol. Chem. 1997; 272: 1703-1708Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Zhang Y. Bendahan A. Zarbiv R. Kavanaugh M.P. Kanner B.I. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 751-755Crossref PubMed Scopus (88) Google Scholar), and glutamate (27Bendahan A. Armon A. Madani N. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 2000; 275: 37436-37442Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 28Teichman S. Kanner B.I. J. Gen. Physiol. 2007; 129: 527-539Crossref PubMed Scopus (32) Google Scholar) are facing toward the binding pocket. Recent studies indicate that glutamate translocation occurs by an “elevator-like” mechanism (29Reyes N. Ginter C. Boudker O. Nature. 2009; 462: 880-885Crossref PubMed Scopus (338) Google Scholar, 30Crisman T.J. Qu S. Kanner B.I. Forrest L.R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 20752-20757Crossref PubMed Scopus (119) Google Scholar) where the transport domain, which includes HP1 and HP2 and TMs 3, 6, 7, and 8, moves relative to the fixed trimerization domain (31Groeneveld M. Slotboom D.J. J. Mol. Biol. 2007; 372: 565-570Crossref PubMed Scopus (54) Google Scholar). transmembrane domain. Because of the limited resolution of the GltPh structure, Tl+ ions, which exhibit a robust anomalous scattering signal, have been used in an attempt to visualize the sodium sites in this homologue (24Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Nature. 2007; 445: 387-393Crossref PubMed Scopus (396) Google Scholar), which also uses 3 Na+ ions per transported substrate molecule (32Groeneveld M. Slotboom D.J. Biochemistry. 2010; 49: 3511-3513Crossref PubMed Scopus (87) Google Scholar). Two Tl+ sites were identified. One was found to be buried just under HP2 and seemed to have only four coordinating main chain carbonyl oxygens from TM7 and HP2. The other Tl+ site was buried deeply within the protein, coordinated by three main chain carbonyl oxygens from TM7 and TM8 as well as the two carboxyl oxygens of a conserved TM8 aspartate residue (Asp-405) and possibly a hydroxyl oxygen of a HP1 serine residue (24Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Nature. 2007; 445: 387-393Crossref PubMed Scopus (396) Google Scholar). There is nevertheless, uncertainty on the assumption that Tl+ faithfully reports on Na+ because in contrast to Na+, Tl+ could not support transport (24Boudker O. Ryan R.M. Yernool D. Shimamoto K. Gouaux E. Nature. 2007; 445: 387-393Crossref PubMed Scopus (396) Google Scholar). However, functional evidence supports the role of one of the Tl+ sites as a sodium binding site (33Teichman S. Qu S. Kanner B.I. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14297-14302Crossref PubMed Scopus (36) Google Scholar). In the absence of high resolution structural data, suggestions for additional sodium binding sites have been searched by using a combination of computational and functional studies (34Tao Z. Rosental N. Kanner B.I. Gameiro A. Mwaura J. Grewer C. J. Biol. Chem. 2010; 285: 17725-17733Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 35Larsson H.P. Wang X. Lev B. Baconguis I. Caplan D.A. Vyleta N.P. Koch H.P. Diez-Sampedro A. Noskov S.Y. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 13912-13917Crossref PubMed Scopus (67) Google Scholar, 36Rosental N. Bendahan A. Kanner B.I. J. Biol. Chem. 2006; 281: 27905-27915Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). It has been proposed that one of these sites (34Tao Z. Rosental N. Kanner B.I. Gameiro A. Mwaura J. Grewer C. J. Biol. Chem. 2010; 285: 17725-17733Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) represents a transient site (35Larsson H.P. Wang X. Lev B. Baconguis I. Caplan D.A. Vyleta N.P. Koch H.P. Diez-Sampedro A. Noskov S.Y. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 13912-13917Crossref PubMed Scopus (67) Google Scholar). This suggests the possibility that the number of sodium sites may be even higher than three. In the past, we have obtained functional evidence that the conserved tyrosine residue, corresponding to Tyr-317 of GltPh, represents a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1 (10Zhang Y. Bendahan A. Zarbiv R. Kavanaugh M.P. Kanner B.I. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 751-755Crossref PubMed Scopus (88) Google Scholar). In the GltPh structure, a conserved aspartate residue (Asp-390) is located adjacent to the conserved tyrosine residue (16Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (668) Google Scholar) (Fig. 1B), suggesting the possibility that these two residues could participate together in the formation of a new and as yet unidentified cation binding site. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of GltPh. We found that only one of the substitution mutants, EAAC1-D440E, exhibited transport activity. The functional characteristics of this mutant are consistent with the possibility that the conserved aspartate and tyrosine residues could be involved in the formation of a novel cation binding site. The C-terminal histidine-tagged versions of rabbit EAAC1 (37Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1198) Google Scholar, 38Borre L. Kanner B.I. J. Biol. Chem. 2004; 279: 2513-2519Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) in the vector pBluescript SK− (Stratagene) was used as a parent for site-directed mutagenesis (39Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 40Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). This was followed by subcloning the mutations into the His-tagged EAAC1, residing in the oocyte expression vector pOG1(38Borre L. Kanner B.I. J. Biol. Chem. 2004; 279: 2513-2519Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), using the unique restriction enzymes NsiI and StuI. The subcloned DNA fragments were sequenced between these restriction sites. HeLa cells were cultured (41Keynan S. Suh Y.J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (122) Google Scholar), infected with the recombinant vaccinia/T7 virus vTF7–3 (42Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 8122-8126Crossref PubMed Scopus (1874) Google Scholar), and transfected with the plasmid DNA harboring the WT or mutant constructs or with the plasmid vectors alone (41Keynan S. Suh Y.J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (122) Google Scholar). Transport of d-[3H]aspartate or other radiolabeled substrates was done as described (39Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, HeLa cells were plated on 24-well plates and washed with transport medium containing 150 mm NaCl, 5 mm potassium Pi, pH 7.4. Each well was then incubated with 200 μl of transport medium supplemented with 0.4 μCi of the tritium-labeled substrates for 10 min, followed by washing, solubilization of the cells with SDS, and scintillation counting. Solubilization of transporters expressed in the HeLa cells, their reconstitution in proteoliposomes, and transport experiments were done as described (6Kavanaugh M.P. Bendahan A. Zerangue N. Zhang Y. Kanner B.I. J. Biol. Chem. 1997; 272: 1703-1708Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 43Pines G. Danbolt N.C. Bjørås M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1136) Google Scholar). Briefly, 10 μl of proteoliposomes were diluted into 360 μl of 150 mm NaCl, supplemented with 1 μCi of d-[3H]aspartate (11.3 Ci/mmol) or l-[3H]aspartate (12.9 Ci/mmol) and 2.5 μm valinomycin for each triplicate time point. For the experiments depicted in FIGURE 7, FIGURE 8, the amount of radioactive substrate was 4 μCi. For the determination of the kinetic parameters for d- and l-aspartate net flux (1 min) the substrate concentration was varied between 0.15 and 6 μm using 0.6 or 4.0 μCi of radioactivity for substrate concentrations below or above 0.5 μm, respectively. Exchange was measured exactly as described (6Kavanaugh M.P. Bendahan A. Zerangue N. Zhang Y. Kanner B.I. J. Biol. Chem. 1997; 272: 1703-1708Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and the data are presented as net exchange after subtracting the values obtained on proteoliposomes containing only 0.12 m sodium Pi, pH 7.4, from those containing 0.12 m sodium Pi + 10 mm l-aspartate. Protein was determined by the Lowry method (44Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Experiments were done at least three times.FIGURE 8Cation selectivity of net flux by wild type and D440E. Net flux of l-[3H]aspartate (l-Asp) or d-[3H]aspartate (d-Asp) mediated by proteoliposomes containing EAAC1-WT (white bars) or EAAC1-D440E (black bars) was performed as described for Fig. 7, B and C, in the presence of 150 mm NaCl (Na+) or 150 mm LiCl (Li+) in the external medium. Uptake values were corrected as above, and normalized separately for each condition to the value of l-[3H]aspartate flux by EAAC1-WT proteoliposomes. Data are mean ± S.E. of three repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) cRNA was transcribed using mMESSAGE-mMACHINE (Ambion), injected into Xenopus laevis oocytes, which were maintained as described (26Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Oocytes were placed in the recording chamber, penetrated with two agarose-cushioned micropipettes (1%/2 m KCl, resistance varied between 0.5 and 3 mΩ), voltage clamped using GeneClamp 500 (Axon Instruments), and digitized using Digidata 1322 (Axon Instruments) both controlled by the pClamp9.0 suite (Axon Instruments). Voltage jumping was performed using a conventional two-electrode voltage clamp as described previously (38Borre L. Kanner B.I. J. Biol. Chem. 2004; 279: 2513-2519Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The standard buffer, termed ND96, was composed of 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm Na-HEPES, pH 7.5. The compositions of other perfusion solutions are indicated in the figure legends. Offset voltages in chloride substitution experiments were avoided by use of an agarose bridge (1%/2 m KCl) that connected the recording chamber to the Ag/AgCl ground electrode. For uptake, four to five oocytes of each mutant were incubated for 20 min in ND96 containing d-[3H]aspartate as described previously (26Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Cell surface biotinylation was done as described previously (36Rosental N. Bendahan A. Kanner B.I. J. Biol. Chem. 2006; 281: 27905-27915Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Briefly, 5 oocytes expressing wild type or mutant EAAC1 were treated with 1.5 mg/ml of sulfosuccinimidyl-2-(biotinamide)ethyl-1,3-dithiopropionate (Pierce) dissolved in ND96 and the streptavidin beads were eluted with a final volume of 70 μl of SDS-PAGE sample buffer. For samples of total cell transporter, 10% of the lysate, before the streptavidin treatment, was run on the same gel as samples eluted from the beads. The Western blots were probed with an affinity purified antibody directed against rabbit EAAT3 (generously provided by N. C. Danbolt, University of Oslo; anti-C491 (Ab,371 (45Holmseth S. Dehnes Y. Bjørnsen L.P. Boulland J.L. Furness D.N. Bergles D. Danbolt N.C. Neuroscience. 2005; 136: 649-660Crossref PubMed Scopus (45) Google Scholar)). Rat EAAC1 cloned from rat retina (15Grewer C. Watzke N. Wiessner M. Rauen T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9706-9711Crossref PubMed Scopus (153) Google Scholar, 46Rauen T. Rothstein J.D. Wässle H. Cell Tissue Res. 1996; 286: 325-336Crossref PubMed Scopus (228) Google Scholar) was subcloned into the vector pBK-CMV (Stratagene) and used for transient transfection of subconfluent human embryonic kidney cell (HEK293; ATCC number CGL 1573) cultures with the calcium phosphate-mediated transfection method, as described (47Chen C. Okayama H. Mol. Cell Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4821) Google Scholar). Electrophysiological recordings were performed 24 h after the transfection for 2 days. The rapid solution exchange (time resolution ∼100–200 ms) was performed by means of a quartz tube (opening 350 μm) positioned at a distance of ≈0.5 mm to the cell. The linear flow rate of the solutions emerging from the opening of the tube was ∼5–10 cm/s. Laser-pulse photolysis experiments were performed as described previously (15Grewer C. Watzke N. Wiessner M. Rauen T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9706-9711Crossref PubMed Scopus (153) Google Scholar). 2 mm 4-methoxy-7-nitroindolinyl-caged glutamate (TOCRIS) was applied to the cells and photolysis of the caged glutamate was initiated with a light flash (355 nm Nd:YAG laser, Minilite series, Continuum). The light was coupled into a quartz fiber (diameter 350 μm) that was positioned in front of the cell at a distance of 300 μm. Laser energies were varied in the 50–300 mJ/cm2 range with neutral density filters. With maximum light intensities of 300 mJ/cm2, saturating glutamate concentrations could be released, which was tested by comparison of the steady-state current with that generated by rapid perfusion of the same cell with 1 mm glutamate. Data were recorded using the pClamp6 software (Axon Instruments), digitized with a sampling rate of 25 kHz and low-pass filtered at 3–10 kHz. All current-voltage relationships represent steady-state substrate-elicited net currents ((Ibuffer+substrate) − (Ibuffer)) and were analyzed by Clampfit version 8.2 or 9.0 (Axon Instruments), and the data have been normalized as indicated in the figure legends. Charge movements were quantitated by integrating the current-time relationships using nonsubtracted or subtracted current records as indicated in the figure legends. Kinetic parameters were determined by nonlinear fitting to the generalized Hill equation using the built-in functions of Origin 6.1 (Microcal). For determination of the apparent affinity for substrate, Imax and K0.5 were allowed to vary and the value of nH was fixed at 1. Origin software was also used to fit the transport current responses to the photolytic release of glutamate. The time constants were obtained by a two-exponential fit of the decaying phases. To test the role of the conserved aspartate residue located at position 440 in EAAC1, we first measured d-[3H]aspartate uptake in HeLa cells expressing mutants where this residue was replaced by glutamate, asparagine, glutamine, serine, or cysteine. As shown in Fig. 2A, none of the mutants tested exhibited significant transport activity, except for D440E, which had activity comparable to that of wild type (EAAC1-WT). Similar results were also obtained by measuring d-[3H]aspartate uptake in oocytes expressing the same mutants (Fig. 2B). To explore the possibility that the lack of transport activity in D440N, D440Q, D440S, and D440C is a consequence of impaired expression at the plasma membrane, we performed cell surface biotinylation in oocytes and found that the biotinylation levels of the Asp-440 mutants were similar or higher than EAAC1-WT (Fig. 2C). The values for the intensity of the transporter bands of the mutants relative to EAAC1-WT (n = 4) were: D440E, 1.46 ± 0.22; D440N, 1.50 ± 0.11; D440S, 1.43 ± 0.03; D440Q, 1.55 ± 0.23 and D440C, 1.96 ± 0.24. The lack of d-[3H]aspartate uptake by the D440N, D440Q, D440S, and D440C mutants could be due to a dramatically increased Km and/or lowered Vmax. To address these possibilities, without the need to use excessive amounts of radioactive substrate, we measured the transport currents in oocytes expressing the Asp-440 mutants in the presence of 96 mm Na+. No measurable currents were induced by any of the three transporter substrates, d-aspartate, l-aspartate, and l-glutamate (tested at concentrations up to 10 mm) in oocytes expressing D440N, D440Q, D440S, or D440C mutants (data not shown). Only the D440E mutant exhibited transport currents induced by each of the three substrates but not by GABA. However, their voltage dependence was remarkably different from those by WT (FIGURE 3, FIGURE 4). The substrate-induced currents (Fig. 3, right panel, l-Asp-Na+) are defined as the currents obtained in the presence of sodium and the substrate (l-aspartate in the case of Fig. 3, middle panel, l-Asp) minus those in sodium without the substrate (Fig. 3, left panel, Na+). When external Na+ was substituted by choline, substrate-induced currents were neither observed in D440E nor in EAAC1-WT (data not shown).FIGURE 4Substrate-induced steady-state currents by EAAC1-WT and D440E. Steady-state currents induced by a saturating concentration (2 mm) of l-aspartate (squares), l-glutamate (circles), d-aspartate (triangles), or GABA (inverted triangles) are shown. Data obtained from oocytes expressing either EAAC1-WT (A) or D440E (B) in a NaCl-based external medium (ND96), as described under “Experimental Procedures,” using the same voltage protocol as described in the legend to Fig. 3. Substrate-induced steady-state currents (Isubstrate − IND96) from 210 to 245 ms at each potential were averaged and normalized to the current induced by l-aspartate at −100 mV (I/Imax). These currents were then plotted against the corresponding membrane potential (Vm (mV)). Data are mean ± S.E. of at least three repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the presence of 96 mm Na+, but in the absence of substrate, transient currents were observed in oocytes expressing EAAC1-WT (Fig. 3, Na+). These currents are proposed to reflect charge-moving conformational changes induced by Na+ binding and unbinding from the empty transporter. Upon substrate application, these transient currents were converted into steady-state currents (Fig. 3, l-Asp). The ratio of the charge moved (derived from the “off”-transient currents; jumping back to the holding potential) in the presence of Na+ (area to the right of the vertical line in inset a) to that in the additional presence of l-aspartate (area to the right of the vertical line in the EAAC1-WT counterpart of inset b) was 9.13 ± 1.68 (n = 3). In contrast, little, if any, transient currents were observed in oocytes expressing the D440E mutant using the same conditions as used in the wild-type (Fig. 3, Na+), but, remarkably, now l-aspartate-induced transient currents (Fig. 3, l-Asp) and the above ratio of the charge moved was 0.25 ± 0.02 for D440E (n = 3). Similar transient currents were also induced by d-aspartate and l-glutamate but not by GABA, which is not a substrate of the glutamate transporters (data not shown). The substrate-induced transient currents by D440E are reminiscent of those observed in previously characterized glutamate transporter mutants, locked in the exchange mode. These transient currents apparently reflect sodium-coupled substrate movement through the membrane electric f" @default.
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• W2046055422 date "2011-12-01" @default.
• W2046055422 modified "2023-10-05" @default.
• W2046055422 title "A Conserved Aspartate Residue Located at the Extracellular End of the Binding Pocket Controls Cation Interactions in Brain Glutamate Transporters" @default.
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• W2046055422 doi "https://doi.org/10.1074/jbc.m111.291021" @default.
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