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• W2000577135 abstract "Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a β-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step. Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a β-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step. Glutamate transporters remove glutamate, the major excitatory neurotransmitter in the brain, from the synaptic cleft and thereby enable the post-synaptic receptors to sense glutamate released from the pre-synaptic nerve terminals. One of the major observations highlighting the importance of these transporters comes from the study of glutamate transporter knockout mice. This study (1Tanaka K. Watase K. Manabe T. Yamada K. Watanabe M. Takahashi K. Iwama H. Nishikawa T. Ichihara N. Kikuchi T. Okuyama S. Kawashima N. Hori S. Takimoto M. Wada K. Science. 1997; 276: 1699-1702Crossref PubMed Scopus (1472) Google Scholar) indicates that glutamate transporters and in particular GLT-1 (2Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1135) Google Scholar), play a central role in preventing both hyperexcitability and excitotoxicity. GLT-1 and the four other eukaryotic glutamate transporters, GLAST-1 (3Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1096) Google Scholar), EAAC1 (4Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1195) Google Scholar), and EAAT-4 and -5 (5Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1012) Google Scholar, 6Arriza J.L. Eliasof S. Kavanaugh M.P. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4155-4160Crossref PubMed Scopus (799) Google Scholar) have an overall identity of around 50%. Glutamate transport is an electrogenic process (7Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar, 8Brew H. Attwell D. Nature. 1987; 327: 707-709Crossref PubMed Scopus (328) Google Scholar, 9Wadiche J.I. Arriza J.L. Amara S.G. Kavanaugh M.P. Neuron. 1995; 14: 1019-1027Abstract Full Text PDF PubMed Scopus (349) Google Scholar), consisting of two distinct half-cycles (Fig. 1A): first, glutamate is co-transported with sodium and hydrogen ions and subsequently the binding sites reorient upon countertransport of potassium (10Kanner B.I. Bendahan A. Biochemistry. 1982; 21: 6327-6330Crossref PubMed Scopus (166) Google Scholar, 11Pines G. Kanner B.I. Biochemistry. 1990; 29: 11209-11214Crossref PubMed Scopus (91) Google Scholar, 12Kavanaugh 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). The stoichiometry of the process has been determined to be 3Na+:1H+:1K+:glutamate (13Zerangue N. Kavanaugh M.P. Nature. 1996; 383: 634-637Crossref PubMed Scopus (706) Google Scholar, 14Levy L.M. Warr O. Attwell D. J. Neurosci. 1998; 18: 9620-9628Crossref PubMed Google Scholar). In addition to the ion-coupled glutamate translocation, glutamate transporters mediate a thermodynamically uncoupled chloride flux activated by two of the molecules they transport, sodium and glutamate (5Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1012) Google Scholar, 15Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (454) Google Scholar). In EAAC1 (also termed EAAT-3), lithium can replace sodium in coupled glutamate uptake but not in its capacity to gate the glutamate-dependent uncoupled anion conductance (16Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and additional studies have reinforced the idea that the conformation gating of the anion conductance is different from that during substrate translocation (17Seal R.P. Shigeri Y. Eliasof S. Leighton B.H. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15324-15329Crossref PubMed Scopus (62) Google Scholar, 18Ryan R.M. Vandenberg R.J. J. Biol. Chem. 2002; 277: 13494-13500Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 19Borre L. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 2002; 277: 13501-13507Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In addition, the uncoupled anion flux can be altered by substituting some of the amino acid residues of transmembrane (TM) 2The abbreviations used are: TM, transmembrane; WT, wild type; GABA, γ-aminobutyric acid.2The abbreviations used are: TM, transmembrane; WT, wild type; GABA, γ-aminobutyric acid. domain 2, without significantly affecting the properties of coupled glutamate translocation (20Ryan R.M. Mitrovic A.D. Vandenberg R.J. J. Biol. Chem. 2004; 279: 20742-20751Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Despite these insights, little is known about the mechanism of glutamate-induced anion permeation, but it has been suggested that glutamate itself may gate the anion permeation (15Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (454) Google Scholar). Glutamate transporters have a non-conventional topology containing two re-entrant loops and two transmembrane domains (7 and 8) in their carboxyl-terminal half (21Grunewald M. Bendahan A. Kanner B.I. Neuron. 1998; 21: 623-632Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 22Slotboom 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, 23Grunewald M. Kanner B.I. J. Biol. Chem. 2000; 275: 9684-9689Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Moreover the two re-entrant loops are in close proximity (24Brocke L. Bendahan A. Grunewald M. Kanner B.I. J. Biol. Chem. 2002; 277: 3985-3992Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The recently solved crystal structure of the glutamate transporter homologue GltPh (25Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (665) Google Scholar) beautifully confirms and refines both the topology and the proximity of the re-entrant loops. Moreover, the equivalent amino acid residues from the eukaryotic glutamate transporters, which are involved in potassium binding (12Kavanaugh 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, 26Zhang 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), sodium specificity (16Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 27Zhang Y. Kanner B.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1710-1715Crossref PubMed Scopus (76) Google Scholar), and the liganding of the γ-carboxyl group of glutamate (28Bendahan 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) are located at the binding pocket of GltPh (25Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (665) Google Scholar). Therefore, the GltPh structure appears to be a good model for the study of its eukaryotic counterparts. The two helical halves of TM7 are separated by an unwound part consisting of a highly conserved 5-amino acid stretch termed the NMDGT motif (Fig. 1B). It has been shown previously by several studies that each residue of this motif is extremely important for the function of the transporter (16Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29Zarbiv R. Grunewald M. Kavanaugh M.P. Kanner B.I. J. Biol. Chem. 1998; 273: 14231-14237Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In the crystal structure of the archaeal homologue, the amino acid residues of this motif are found to form part of the substrate binding pocket (25Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (665) Google Scholar). The side chains of the methionine and the threonine point toward it; but surprisingly, the side chains of the asparagine and aspartate of this motif are pointing away from the binding pocket. These two residues are seen to interact with each other and with additional residues of TM domains 3, 6, and 8. These interactions were suggested to be important for shaping and stabilizing the binding pocket structure (25Yernool D. Boudker O. Jin Y. Gouaux E. Nature. 2004; 431: 811-818Crossref PubMed Scopus (665) Google Scholar). In this study, we test the hypothesis that interaction of the corresponding asparagine and aspartate residues is important for the function of EAAC1. Our results suggest that besides shaping the substrate binding pocket, asparagine 366 and aspartate 368 of EAAC1 have additional functions in the transport process and are crucial for the execution of the potassium-translocation limb of the transport cycle. Generation and Subcloning of Mutants—The rabbit glutamate transporter EAAC1 (4Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1195) Google Scholar) with 10 histidines added immediately after the open reading frame followed by the stop codon (30Borre 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 as described previously (31Pines G. Zhang Y. Kanner B.I. J. Biol. Chem. 1995; 270: 17093-17097Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 32Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). Restriction enzymes PinAI and PflMI were used to subclone the mutations into the construct containing the His-tagged WT EAAC1 residing in pOG1. The latter is an oocyte expression vector containing a 5′-untranslated Xenopus β-globin sequence and a 3′-poly(A) signal. This EAAC1 construct is termed WT in this study. The subcloned DNA fragments were sequenced on both strands between the two restriction sites noted above. In the case of the N396D/D398N-GLT-1 double mutant, the enzymes BsrGI and PshAI were used for subcloning. With the exception of Fig. 5, all figures document results with the mutant in the EAAC-1 background. In Fig. 5, the activity of the N396D/D398N-GLT-1 double mutant was compared with that of wild type GLT-1. Expression in Oocytes— cRNA was transcribed using mMESSAGE-mMACHINE (Ambion), injected into Xenopus laevis oocytes, and maintained as described previously (16Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Surface Biotinylation—This was done as described previously (33Bennett E.R. Su H. Kanner B.I. J. Biol. Chem. 2000; 275: 34106-34113Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), except that 4–5 oocytes, expressing wild type or mutant EAAC1, were treated with 1.5 mg/ml of sulfosuccinimidyl-2-(biotinamide)ethyl-1,3-dithiopropionate (Pierce) in 2 ml of 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 was not treated with the streptavidin beads and these samples were run on the same gel alongside those eluted from the beads. The Western blots were probed with an affinity purified antibody directed against a peptide corresponding to amino acids 491–523 of rabbit EAAT3 (generously provided by N. C. Danbolt, University of Oslo; anti-C491 (Ab,371 (34Holmseth S. Dehnes Y. Bjornsen L.P. Boulland J.L. Furness D.N. Bergles D. Danbolt N.C. Neuroscience. 2005; 136: 649-660Crossref PubMed Scopus (45) Google Scholar)). Oocyte Electrophysiology and Uptake—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 preformed using a conventional two-electrode voltage clamp as described previously (30Borre 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). In sodium substitution experiments NaCl was replaced by an equimolar concentration of either LiCl or choline Cl. In sodium titration experiments the buffer was composed of 130 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm Tris-HEPES, pH 7.5, and NaCl was replaced by equimolar concentrations of choline Cl. Offset voltages in chloride substitution experiments were avoided by the use of an agarose bridge (2%/2 m KCl) that connected the recording chamber to the Ag/AgCl ground electrode. For the determination of anion selectivity (Fig. 10), 10 mm NaCl of the perfusion solution was replaced isoosmotically by the sodium salt of the tested anions. Voltage jumps in the presence of each of the tested anions (in the presence and absence of l-aspartate) was followed by a ∼20-min washout with ND96 solution and the induced currents in chloride medium were measured again to ensure that the test anion was removed from the oocyte before testing the next anion. For uptake, four to five oocytes of each mutant were incubated for 20 min in ND96 containing d-[2,3-3H]aspartic acid as described previously (16Borre L. Kanner B.I. J. Biol. Chem. 2001; 276: 40396-40401Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Data Analysis—All current-voltage relations represent steady-state net currents ((Ibuffer+AA) – (Ibuffer)) elicited by amino acids (AA) and were analyzed by Clampfit version 8.2 or 9.0 (Axon instruments). Even though there was a large variation in the absolute values of the substrate-induced currents between oocytes of different batches and sometimes even within a given batch, the voltage dependence of these currents as well as their kinetic parameters were always very similar when different oocytes were compared. Therefore the data are presented as normalized currents, usually to those at –100 mV, as indicated in the figure legends. Moreover the range of the values of the absolute currents is also stated in the figure legends. Kinetic parameters were determined by non-linear fitting to the generalized Hill equation using the build-in functions of Origin 6.1 (Microcal). For the determination of the apparent affinity for sodium, Imax, K0.5, and nH were allowed to vary, and for the determination of the apparent affinity for acidic amino acids, the value of nH was fixed to 1. The time constants were estimated using the decay of the charge movements induced by amino acids during the “on” phase using non-linear fitting to the first order decay exponential function of Clampfit 8.2 (Axon instruments). Cell Growth and Expression—HeLa cells were cultured (35Keynan S. Suh Y.J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (122) Google Scholar), infected with recombinant vaccinia/T7 virus vTF7–3 (36Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1872) Google Scholar), and transfected with plasmid DNA encoding WT-GLT-1, N396D/D398N-GLT-1, or the vector pBluescript SK– alone, as described (35Keynan S. Suh Y.J. Kanner B.I. Rudnick G. Biochemistry. 1992; 31: 1974-1979Crossref PubMed Scopus (122) Google Scholar). Solubilization of transporters expressed in the HeLa cells, their reconstitution in proteoliposomes (2Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1135) Google Scholar, 12Kavanaugh 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 transport experiments (2Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1135) Google Scholar, 12Kavanaugh 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) were done as described. Exchange data are presented as net exchange, after subtracting the values obtained on proteoliposomes not containing 10 mml-aspartate but only 0.12 m sodium phosphate, pH 7.4. Protein was determined by Lowry's method (37Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Substrate Interactions in N366Q and D368E Mutants—In contrast to oocytes expressing wild type (WT) EAAC1, where a saturating concentration of each of the three substrates, l-aspartate, d-aspartate, and l-glutamate (2 mm), induced currents of similar size (Fig. 2A), this was not the case in substitution mutants N366Q and D368E, where currents induced by 10 mm d-aspartate or l-glutamate were much smaller than those induced by the same concentration of l-aspartate (Fig. 2, B and C). In the N366Q mutant the Imax value for d-aspartate was only 39 ± 1% of that for l-aspartate. In the case of l-glutamate, even at 20 mm saturation was not yet reached (data not shown). In WT EAAC1, the Km values for the three substrates (at –100 mV) were around 10 μm (Table 1). In N366Q, the Km values for l-aspartate, d-aspartate, and l-glutamate were higher than WT EAAC1 by around 100-, 350-, and 1000-fold, respectively (Table 1). In the case of the D368E mutant the increase of the Km for l-aspartate was not as dramatic as in N366Q, around 20-fold. On the other hand l-glutamate-induced currents were too small for quantitative analysis and even at 10 mm the induced inward currents at –100 mV were smaller than 30 nA. In the case of d-aspartate, the Km values of the two mutants were similar, around 2.5 mm (Table 1).TABLE 1The apparent affinity for acidic amino acid of the EAAC1 mutantsl-Aspl-Glud-AspWT0.015 ± 0.0020.013 ± 0.0010.008 ± 0.007N366Q1.66 ± 0.3412.6 ± 0.762.90 ± 0.17D368E0.32 ± 0.05—aValues of l-glutamate-induced currents in D368E were too low for apparent affinity determination.2.40 ± 0.28ND/DN0.073 ± 0.020.25 ± 0.080.067 ± 0.032WT, low Na+0.053 ± 0.0140.065 ± 0.0130.039 ± 0.007a Values of l-glutamate-induced currents in D368E were too low for apparent affinity determination. Open table in a new tab No uptake of d-[2,3-3H]aspartate by the N366Q and D368E mutants could be detected (Fig. 3) and the same was true for the uptake of l-[2,3-3H]aspartate (data not shown), apparently because of the dramatic increase of the Km values of the mutants for the acidic amino acids (Table 1). Surface biotinylation using an anti-EAAC1 antibody showed that the lower functional expression of N366Q and D368E was not due to lower expression of these transporters on the plasma membrane than the WT (Fig. 4). In the samples of the total protein of wild type and mutants, bands of around 55–60 and 65–70 kDa were observed, which apparently represent non-mature and mature monomeric forms of the transporter, respectively. Moreover bands of lower mobility, apparently representing aggregated transporters, were observed (Fig. 4), although in other experiments the monomeric form was more abundant (data not shown). In the biotinylated samples, representing those transporters located at the plasma membrane, the mature form was predominant among the monomeric transporters, except for the case of N366Q (Fig. 4). In other experiments the pattern of N366Q was not different from that of the other mutants (data not shown). The specificity of the bands is illustrated by the fact that the transporter bands were not observed in total and biotinylated samples of non-injected oocytes (uninj., Fig. 4). In the depicted experiment, the mutant transporters were present on the plasma membrane at similar or higher levels than those of WT (Fig. 4). Even though there was variation between experiments, the expression of the mutants was usually at least that of WT (data not shown). The proportion of monomeric to aggregated transporter also changed between experiments, such that sometimes the low-mobility bands were predominant (data not shown). It was difficult to control this variability, which is apparently related to the well known property of glutamate transporters to aggregate (38Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (369) Google Scholar). Nevertheless, whatever the aggregation state of the transporter, these bands were never seen with uninjected oocytes, just as shown in Fig. 4.FIGURE 4Cell surface biotinylation of the wild type and mutants. Oocytes expressing EAAC1-WT and the indicated mutants, were labeled and processed as described under “Experimental Procedures.” The six left lanes show the total samples, and the six right lanes show the biotinylated samples. The first lane of each group shows a sample from uninjected oocytes (Uninj.). The empty lane between the two groups contained the marker proteins and their positions (in kDa) are indicated on the left. All samples were separated on the same SDS gel, transferred to nitrocellulose, and detected as described under “Experimental Procedures.” Shown is a representative of four separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the N366D, D368N, N366A, and D368A mutants, none of the three substrates (tested at concentrations up to 10 mm) induced any measurable currents (data not shown) and no uptake of d-[2,3-3H]aspartate was observed (Fig. 3). Again, as found by surface biotinylation, the lack of functional expression of these four mutants is not due to lower expression of these transporters on the plasma membrane (Fig. 4 and data not shown). As will be shown below, the N366Q had a lowered apparent affinity for sodium. However, lowering the external sodium concentration from 100 to 20 mm had only a relatively modest effect on the Km values for the three substrates in WT EAAC1 as compared with the impact of the mutations (Table 1). Thus it appears that the dramatic increase in Km for the three substrates in N366Q is not just a consequence of its reduced apparent affinity for sodium. Radioactive Uptake by N366/D368 Double Mutants—In contrast to the single mutants at positions 366 and 368, oocytes expressing the N366D/D368N double mutant exhibited significant, albeit slow, uptake of d-[3H]aspartate (Fig. 3). As also shown in Fig. 3, such uptake was neither observed when the neutral alanine was introduced at both positions (N366A/D368A) nor in the N366Q/D366E and N366E/D366Q double mutants. Also in the case of the double mutants, we found that their expression level at the plasma membrane was similar to that of WT (data not shown). The observation of radioactive substrate uptake in the N366D/D368N mutant, which does not exhibit steady-state transport currents (see Figs. 6 and 7) is reminiscent of the E404D mutant of the glutamate transporter GLT-1, which has an impaired imteraction with potassium and is therefore locked in the exchange mode (12Kavanaugh 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). Because oocytes have high (around 12 mm) intracellular levels of acidic amino acids (13Zerangue N. Kavanaugh M.P. Nature. 1996; 383: 634-637Crossref PubMed Scopus (706) Google Scholar), oocytes expressing the E404D mutant are able to take up radioactive acidic amino acids (in exchange for their endogenous unlabeled counterparts) to almost the same levels as those expressing WT (12Kavanaugh 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). One way to test if the N366D/D368N mutant is locked in the exchange mode is to use reconstituted systems using the transporters expressed in cell lines, such as HeLa cells. Because we anticipated low exchange levels in the double mutant, we used the equivalent N396D/D398N double mutant in the background of the glutamate transporter GLT-1, which is much more robustly expressed in the heterologous HeLa cell expression system than EAAC1. Under net flux conditions, created by dilution of potassium containing liposomes, inlaid with wild type GLT-1, into a sodium-containing transport medium in the presence of the potassium-specific ionophore valinomycin (2.5 μm), d-[2,3-3H]aspartate uptake was observed (Fig. 5, net flux). Such uptake was not seen when the liposomes were inlaid with similarly solubilized proteins from HeLa cells transfected with the vector alone (SK), whereas with N396D/D398N double mutant liposomes (ND/DN) net flux amounted for no more than 2% of that of WT (Fig. 5). On the other hand, much higher d-[3H]aspartate uptake was seen in sodium and l-aspartate (10 mm) containing N396D/D398N liposomes (increment over similar liposomes containing sodium without l-aspartate), even though exchange by wild type GLT-I liposomes was more robust (Fig. 5, exchange). In the control liposomes (from cells transfected with the vector alone), very little exchange was observed (Fig. 5). As observed previously in GLT-1 liposomes (12Kavanaugh 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), uptake was also seen in N396D/D398N liposomes containing l-glutamate, but not in those containing γ-aminobutyric acid (GABA), which is not a substrate of the glutamate transporters (data not shown). Similar results were also obtained when uptake of l-[2,3-3H]aspartate was tested (Fig. 5, exchange).FIGURE 7Cation dependence of l-aspartate-induced transient currents by the N366D/D368N mutant. Currents induced by l-aspartate (300 μm) were recorded using the same voltage protocol described in the legend to Fig. 6 in ND96 (A, Na+). No response was observed upon l-aspartate application when sodium was substituted by an equimolar concentration of either lithium (B, Li+) or choline (data not shown, n = 5). The dashed lines indicate 0 current. The data shown in A and B are from the same oocyte and are representative of five oocytes.View Large" @default.
• W2000577135 created "2016-06-24" @default.
• W2000577135 creator A5038910133 @default.
• W2000577135 creator A5041387956 @default.
• W2000577135 creator A5060349010 @default.
• W2000577135 date "2006-09-01" @default.
• W2000577135 modified "2023-10-03" @default.
• W2000577135 title "Multiple Consequences of Mutating Two Conserved β-Bridge Forming Residues in the Translocation Cycle of a Neuronal Glutamate Transporter" @default.
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