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• W2083347740 abstract "The neuronal transporter excitatory amino acid carrier 1 (EAAC1) is enriched in perisynaptic regions, where it may regulate synaptic spillover of glutamate. In this study we examined potential interactions between EAAC1 and ionotropic glutamate receptors. N-Methyl-d-aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B, but not the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR2, were co-immunoprecipitated with EAAC1 from neuron-enriched hippocampal cultures. A similar interaction was observed in C6 glioma and human embryonic kidney cells after co-transfection with Myc epitope-tagged EAAC1 and NMDA receptor subunits. Co-transfection of C6 glioma with the combination of NR1 and NR2 subunits dramatically increased (∼3-fold) the amount of Myc-EAAC1 that can be labeled with a membrane-impermeable biotinylating reagent. In hippocampal cultures, brief (5 min), robust (100 μm NMDA, 10 μm glycine) activation of the NMDA receptor decreased biotinylated EAAC1 to ∼50% of control levels. This effect was inhibited by an NMDA receptor antagonist, intracellular or extracellular calcium chelators, or hypertonic sucrose. Glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with cyclothiazide, and thapsigargin mimicked the effects of NMDA. These studies suggest that NMDA receptors interact with EAAC1, facilitate cell surface expression of EAAC1 under basal conditions, and control internalization of EAAC1 upon activation. This NMDA receptor-dependent regulation of EAAC1 provides a novel mechanism that may shape excitatory signaling during synaptic plasticity and/or excitotoxicity. The neuronal transporter excitatory amino acid carrier 1 (EAAC1) is enriched in perisynaptic regions, where it may regulate synaptic spillover of glutamate. In this study we examined potential interactions between EAAC1 and ionotropic glutamate receptors. N-Methyl-d-aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B, but not the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR2, were co-immunoprecipitated with EAAC1 from neuron-enriched hippocampal cultures. A similar interaction was observed in C6 glioma and human embryonic kidney cells after co-transfection with Myc epitope-tagged EAAC1 and NMDA receptor subunits. Co-transfection of C6 glioma with the combination of NR1 and NR2 subunits dramatically increased (∼3-fold) the amount of Myc-EAAC1 that can be labeled with a membrane-impermeable biotinylating reagent. In hippocampal cultures, brief (5 min), robust (100 μm NMDA, 10 μm glycine) activation of the NMDA receptor decreased biotinylated EAAC1 to ∼50% of control levels. This effect was inhibited by an NMDA receptor antagonist, intracellular or extracellular calcium chelators, or hypertonic sucrose. Glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with cyclothiazide, and thapsigargin mimicked the effects of NMDA. These studies suggest that NMDA receptors interact with EAAC1, facilitate cell surface expression of EAAC1 under basal conditions, and control internalization of EAAC1 upon activation. This NMDA receptor-dependent regulation of EAAC1 provides a novel mechanism that may shape excitatory signaling during synaptic plasticity and/or excitotoxicity. Glutamate is an excitatory amino acid that elicits physiological and excitotoxic responses in the nervous system. Extracellular glutamate is normally maintained at low levels, allowing bursts of glutamate to generate postsynaptic responses during synaptic transmission. Tight regulation of extracellular glutamate is required to allow essential activity and prevent excitotoxicity (for reviews, see Refs. 1Danbolt N.C. Prog. Neurobiol. (N. Y.). 2001; 65: 1-105Crossref PubMed Scopus (3701) Google Scholar, 2Amara S.G. Sonders M.S. Zahniser N.R. Povlock S.L. Daniels G.M. Adv. Pharmacol. 1998; 42: 164-168Crossref PubMed Scopus (39) Google Scholar, 3Robinson M.B. Neurochem. Int. 1999; 33: 479-491Crossref Scopus (21) Google Scholar).Metabotropic (mGluR) 3The abbreviations used are: mGluR, metabotropic glutamate receptor; DIV, days in vitro; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-d-aspartate; EAAC1, excitatory amino acid carrier 1; LTP, long-term potentiation; LTD, long-term depression; BisII, bisindolmaleimide II; d-APV, d-2-amino-5-phosphonovalerate; CMV, cytomegalovirus. 3The abbreviations used are: mGluR, metabotropic glutamate receptor; DIV, days in vitro; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; NMDA, N-methyl-d-aspartate; EAAC1, excitatory amino acid carrier 1; LTP, long-term potentiation; LTD, long-term depression; BisII, bisindolmaleimide II; d-APV, d-2-amino-5-phosphonovalerate; CMV, cytomegalovirus. and ionotropic (AMPA, kainate, and NMDA) receptors mediate the effects of glutamate. mGluRs are G-protein-coupled receptors that activate a variety of second-messenger systems (for review, see Ref. 4Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2704) Google Scholar), whereas AMPA, kainate, and NMDA receptors are ligand-gated ion channels that also activate intracellular signaling pathways. NMDA receptors have a high affinity for glutamate, are highly permeable to calcium, and do not readily desensitize, all of which contribute to the central role of NMDA receptors in excitotoxicity (5Sattler R. Tymianski M. Mol. Neurobiol. 2001; 24: 107-129Crossref PubMed Scopus (458) Google Scholar, 6Waxman E.A. Lynch D.R. Neuroscientist. 2005; 11: 37-49Crossref PubMed Scopus (298) Google Scholar).Removal of extracellular glutamate in the forebrain is controlled by three major excitatory amino acid transporters (EAATs): EAAT1 (GLAST), EAAT2 (GLT-1), and EAAT3 (EAAC1) (for reviews, see Refs. 1Danbolt N.C. Prog. Neurobiol. (N. Y.). 2001; 65: 1-105Crossref PubMed Scopus (3701) Google Scholar and 7Sims K.D. Robinson M.B. Crit. Rev. Neurobiol. 1999; 13: 169-197Crossref PubMed Scopus (146) Google Scholar). Glutamate transporters rapidly clear extracellular glutamate, thereby protecting neurons from excitotoxicity (for reviews, see Refs. 1Danbolt N.C. Prog. Neurobiol. (N. Y.). 2001; 65: 1-105Crossref PubMed Scopus (3701) Google Scholar, 2Amara S.G. Sonders M.S. Zahniser N.R. Povlock S.L. Daniels G.M. Adv. Pharmacol. 1998; 42: 164-168Crossref PubMed Scopus (39) Google Scholar, 3Robinson M.B. Neurochem. Int. 1999; 33: 479-491Crossref Scopus (21) Google Scholar). Although GLAST and GLT-1 are mainly glial, EAAC1 is mostly neuronal (8Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1448) Google Scholar). In area CA1 of the hippocampus, a neuronal transporter limits synaptic spillover of glutamate and activation of NMDA receptors (9Diamond J.S. J. Neurosci. 2001; 21: 8328-8338Crossref PubMed Google Scholar). Because EAAC1 is the only neuronal transporter found in this location, this observation suggests that EAAC1 may control activation of some subtypes of NMDA receptors. A similar functional interaction is also observed in Xenopus oocytes, where co-expression of EAAC1 attenuates glutamate-evoked NMDA receptor currents (10Zuo Z. Fang H. J. Exp. Biol. 2005; 208: 2063-2070Crossref PubMed Scopus (8) Google Scholar). EAAC1 has also been linked to pathology in the nervous system. For example, antisense-mediated down-regulation of EAAC1 causes glutamate-induced cell death in the hippocampus (11Brustovetsky T. Purl K. Young A. Shimizu K. Dubinsky J.M. Exp. Neurol. 2004; 189: 222-230Crossref PubMed Scopus (29) Google Scholar, 12Rothstein J.D. Dykes-Hoberg M. Pardo C.A. Bristol L.A. Jin L. Kuncl R.W. Kanai Y. Hediger M. Wang Y. Schielke J.P. Welty D.F. Neuron. 1996; 16: 675-686Abstract Full Text Full Text PDF PubMed Scopus (2110) Google Scholar). However, some studies suggest that glutamate transporters contribute to excitotoxicity through transporter reversal during insults such as ischemia (13Rossi D.J. Oshima T. Attwell D. Nature. 2000; 403: 316-321Crossref PubMed Scopus (1199) Google Scholar, 14Roettger V. Lipton P. Neuroscience. 1996; 75: 677-685Crossref PubMed Scopus (90) Google Scholar). Based on these studies, EAAC1 may play important roles in NMDA receptor-mediated excitotoxicity.Whereas NMDA receptors are localized to presynaptic, synaptic, and extrasynaptic areas (15Janssen W.G. Vissavajjhala P. Andrews G. Moran T. Hof P.R. Morrison J.H. Exp. Neurol. 2005; 191: S28-S44Crossref PubMed Scopus (50) Google Scholar), EAAC1 is enriched perisynaptically (16He Y. Hof P.H. Janssen W.G.M. Rothstein J.D. Morrison J.H. Neurosci. Lett. 2001; 311: 161-164Crossref PubMed Scopus (27) Google Scholar, 17He Y. Janssen W.G.M. Rothstein J.D. Morrison J.H. J. Comp. Neurol. 2000; 418: 255-269Crossref PubMed Scopus (124) Google Scholar), placing EAAC1 in close proximity to subpopulations of NMDA receptors. NMDA receptor activation can mediate both long-term potentiation (LTP) and long-term depression (LTD) (18Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1319) Google Scholar, 19Massey P.V. Johnson B.E. Moult P.R. Auberson Y.P. Brown M.W. Molnar E. Collingridge G.L. Bashir Z.I. J. Neurosci. 2004; 24: 7821-7828Crossref PubMed Scopus (560) Google Scholar). Recent preliminary data presented in an abstract suggest that knockdown of EAAC1 may impair NMDA receptor-induced LTP (20Beckman M.L. Leary G. Awes A.N. Esslinger C.S. Poulsen D. Babcock M. Manzoni O. Kavanaugh M.P. 35th Annual Meeting of the Society for Neuroscience, November 12–16, Washington, D. C., Abstr. 38.15. Society for Neuroscience, Washington, D. C.2005Google Scholar). EAAC1 activity may, therefore, influence NMDA receptor function. Conversely, LTP increases glutamate uptake activity, which has pharmacological properties consistent with EAAC1 (21Levenson J. Weeber E. Selcher J.C. Kategaya L.S. Sweatt J.D. Eskin A. Nat. Neurosci. 2002; 5: 155-161Crossref PubMed Scopus (124) Google Scholar). This effect is associated with an increase in EAAC1 immunoreactivity in a subcellular fraction enriched in plasma membranes and is blocked by NMDA receptor antagonists (21Levenson J. Weeber E. Selcher J.C. Kategaya L.S. Sweatt J.D. Eskin A. Nat. Neurosci. 2002; 5: 155-161Crossref PubMed Scopus (124) Google Scholar). Therefore, EAAC1 and ionotropic glutamate receptors may be regulated by similar mechanisms that are normally controlled by NMDA receptor stimulation.In this study we sought to identify associations between ionotropic glutamate receptors and EAAC1. We found evidence for physical (co-immunoprecipitable) interactions between EAAC1 and the NMDA receptor in a variety of systems. In addition, co-expression of NMDA receptors with an epitope-tagged variant of EAAC1 increased the amount of biotinylated (cell surface) EAAC1 immunoreactivity. Finally, robust activation of the NMDA receptor rapidly (within 5 min) decreased the amount of biotinylated EAAC1 in hippocampal neuron-enriched cultures. This effect was blocked by chelation of intracellular or extracellular calcium or by hypertonic sucrose. These studies identify novel physical and functional interactions between NMDA receptors and the neuronal transporter EAAC1.EXPERIMENTAL PROCEDURESHippocampal Primary Culture—Neuronally enriched cultures were prepared from the hippocampus of 18–19-day Sprague-Dawley rat embryos (E18-E19) as described (22Wilcox K.S. Buchhalter J. Dichter M.A. Synapse. 1994; 18: 128-151Crossref PubMed Scopus (72) Google Scholar). Briefly, the hippocampi from rat embryos were trypsinized (0.027%) for 20 min at 37 °C in 7% CO2 and washed with Hanks' buffered saline solution. The tissue was triturated in warm media (10% defined, heat-inactivated fetal bovine serum, 10% Ham-s F-12 medium, and 80% Dulbecco's modified Eagle's medium with penicillin/streptomycin), and cells were plated onto 60-mm poly-d-lysine-coated plates at a density of 6 × 105 viable cells per 35-mm culture dish. Neurons were maintained at 37 °C, 5% CO2 with neurobasal media containing B27 (2%). Cultures, which include a heterogeneous mixture of pyramidal and non-pyramidal hippocampal neurons and a small percentage of astrocytes (generally <10%), were maintained for at least 17 days in vitro (DIV17) before experimentation unless otherwise indicated. In some studies cultures were maintained for shorter periods of time (8–9 days, DIV8–9) to model a less “mature” phenotype of synaptic development. At this earlier time, NMDA receptors are primarily composed of the NR1 and NR2B subunits with minimal levels of NR2A and minimal levels of the scaffolding protein PSD-95; this profile is similar to that observed early in development in vivo (23Tovar K.R. Westbrook G.L. J. Neurosci. 1999; 19: 4180-4188Crossref PubMed Google Scholar, 24O'Donnell L.A. Agrawal A. Jordan-Sciutto K.L. Dichter M.A. Lynch D.R. Kolson D.L. J. Neurosci. 2006; 26: 3981-3990Crossref PubMed Scopus (86) Google Scholar, 25Dong Y.N. Wu H.Y. Hsu F. Coulter D.A. Lynch D.R. J. Neurochem. 2006; 99: 206-217Crossref PubMed Scopus (34) Google Scholar). Neuronal cultures maintained for 17 days express combinations of NMDA receptor subunits (NR1, NR2A, and NR2B) and scaffolding proteins (such as PSD-95) similar to those observed in adult animals and, therefore, may model the more mature synaptic milieu.Cell Culture—The rat C6 glioma cell line was grown and maintained as described (26Sheldon A.L. Gonzalez M.I. Robinson M.B. J. Biol. Chem. 2006; 281: 4876-4886Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Under normal conditions this cell line only expresses the EAAC1 subtype of glutamate transporter (27Palos T.P. Ramachandran B. Boado R. Howard B.D. Mol. Brain Res. 1996; 37: 297-303Crossref PubMed Scopus (68) Google Scholar, 28Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar, 29Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (115) Google Scholar). We and others have used this cell line to study EAAC1 trafficking (28Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar, 29Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (115) Google Scholar, 30Najimi M. Maloteaux J.-M. Hermans E. FEBS Lett. 2002; 523: 224-228Crossref PubMed Scopus (31) Google Scholar, 31Najimi M. Maloteaux J.-M. Hermans E. Biochim. Biophys. Acta. 2005; 1668: 195-202Crossref PubMed Scopus (13) Google Scholar). This system has an advantage of being relatively easy to transfect. In addition, the effects of protein kinase C activation, platelet-derived growth factor, and the constitutive trafficking of EAAC1 on and off the plasma membrane display similar characteristics to those observed in neuronal cultures (32Gonzaélez M.I. Kazanietz M.G. Robinson M.B. Mol. Pharmacol. 2002; 62: 901-910Crossref PubMed Scopus (90) Google Scholar, 33Fournier K.M. Gonzaélez M.I. Robinson M.B. J. Biol. Chem. 2004; 279: 34505-34513Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 34Guillet B.A. Velly L.J. Canolle B. Masmejean F.M. Nieoullon A.L. Pisano P. Neurochem. Int. 2005; 46: 337-346Crossref PubMed Scopus (97) Google Scholar). Therefore, this system is analogous to differentiated NIH3T3 cells routinely used to study regulated trafficking of the GLUT4 subtype of glucose transporter (35Martin S. Slot J.W. James D.E. Cell Biochem. Biophys. 1999; 30: 89-113Crossref PubMed Scopus (21) Google Scholar).Incubation in NMDA Receptor Agonists and Selective Antagonists—Agonists and antagonists were acquired from Sigma-Aldrich unless otherwise indicated. To activate NMDA receptors, NMDA (10 or 100 μm) and glycine (10 μm) were diluted to a 5× concentration in conditioned neurobasal media with or without applicable inhibitors and then added directly to culture media for 5 min. Other treatments included glutamate (100 μm) with 10 μm glycine, thapsigargin (200 nm), AMPA (100 μm), cyclothiazide (50 μm), dihydroxyphenylglycine (250 μm), or bisindolmaleimide II (BisII; 10 μm). Cultures were preincubated with inhibitors for 30 min before treatment unless otherwise indicated. Compounds tested as potential blockers of the effects included d-2-amino-5-phosphonovalerate (d-APV; 1 mm), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM; 50 μm), ethylene glycol EGTA (2 mm, 1 min pretreatment), W-7 (5 μm), FK506 (1 μm; a gift from Dr. Ted Dawson, Johns Hopkins University), KN62 (10 μm), 7-nitroindazole (100 μm), MDL28170 (10 μm), MG132 (10 μm; Calbiochem), LY294002 (10 μm), wortmannin (100 nm), SB203580 (1 μm), PD98059 (50 μm), dantrolene (10 μm), SKF-96365 (25 μm), 2-aminoethoxydiphenyl borate (100 μm, 15 min pretreatment; Calbiochem), Ro25-6981 (10 μm), and sucrose (0.45 m). A mixture of voltage-dependent calcium channel inhibitors was also used, including nifedipine (2 μm), ω-agatoxin IVa (30 nm), and ω-conotoxin GVIA (100 nm). BAPTA-AM, W-7, FK506, KN62, 7-nitroindazole, MDL28170, MG132, SB203580, PD98059, and 2-aminoethoxydiphenyl borate were dissolved in Me2SO, and experiments involving these inhibitors were compared with a 0.1% Me2SO vehicle control. Although dantrolene, nifedipine, ω-agatoxin IVa, ω-conotoxin GVIA, LY294002, and wortmannin were also dissolved in Me2SO, the highest concentration of Me2SO for these experiments was 0.02%, which did not affect EAAC1 distribution. Cultures were maintained at 37 °C and 5% CO2 during preincubation and treatment.Transient Transfection of C6 Glioma—C6 glioma were grown to 40–60% confluency on 35- or 60-mm plates and transfected using GenePorter (Genlantis, San Diego, CA), a lipid-based transfection kit. 35-mm plates were transfected with 3–4 μg of cDNA, and 60-mm plates were transfected with 5–7 μg of cDNA with a 5:1 ratio of GenePorter reagent to cDNA, diluted in Dulbecco's modified Eagle's medium as per the manufacturer's recommendations. Cultures were maintained for 18 h after transfection. All transfections included cDNAs for NR2A, NR2B (∈2), and Myc-EAAC1. This combination of cDNAs was co-transfected with either NR1 or pRC/CMV (control vector). During and after transfection, C6 glioma cells were incubated in Dulbecco's modified Eagle's medium with 500 μm ketamine, a concentration that prevents tonic toxic activation of NMDA receptors and consequent excitotoxicity (36Anegawa N.J. Lynch D.R. Verdoorn T.A. Pritchett D.B. J. Neurochem. 1995; 64: 2004-2012Crossref PubMed Scopus (82) Google Scholar, 37Grant E.R. Bacskai B.J. Pleasure D.E. Pritchett D.B. Gallagher M.J. Kendrick S.J. Kricka L.J. Lynch D.R. J. Biol. Chem. 1997; 272: 647-656Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Ketamine was included in all parallel comparison groups to exclude the possibility of nonspecific effects of ketamine with targets unrelated to NMDA receptors. Experiments were standardized for expression levels of Myc-EAAC1. In 2 of 5 experiments Myc-EAAC1 immunoreactivity in the lysate fraction was barely detectable; these Western blots were not quantitated.Transfection of HEK293 Cells—HEK293 cells were grown to 50–70% confluency and transfected using calcium phosphate precipitation (37Grant E.R. Bacskai B.J. Pleasure D.E. Pritchett D.B. Gallagher M.J. Kendrick S.J. Kricka L.J. Lynch D.R. J. Biol. Chem. 1997; 272: 647-656Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). cDNA combinations including Myc-EAAC1, NR1, NR2A, NR2B (∈2), NMDA receptor subunit deletions, and/or pRC/CMV were transfected with a total of 3 μg of cDNA per 35-mm plate. Experiments contained equal total amounts of cDNA. During and after transfection, HEK293 cells were incubated in minimum essential medium with 500 μm ketamine. MK-801 was not used to prevent toxicity because of the slow off-rate. d-APV was not used because in the presence of serum, high concentrations do not completely block toxicity, and the experiments can become quite expensive (for discussion, see Refs. 35Martin S. Slot J.W. James D.E. Cell Biochem. Biophys. 1999; 30: 89-113Crossref PubMed Scopus (21) Google Scholar, 36Anegawa N.J. Lynch D.R. Verdoorn T.A. Pritchett D.B. J. Neurochem. 1995; 64: 2004-2012Crossref PubMed Scopus (82) Google Scholar, 37Grant E.R. Bacskai B.J. Pleasure D.E. Pritchett D.B. Gallagher M.J. Kendrick S.J. Kricka L.J. Lynch D.R. J. Biol. Chem. 1997; 272: 647-656Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Cultures were maintained for 18 h after transfection before experimentation.Co-immunoprecipitation—Cells were washed with ice-cold phosphate-buffered saline solution and incubated in radioimmune precipitation assay buffer (150 mm NaCl, 1 mm EDTA, 100 mm Tris-Cl, 1% Triton X-100, 0.5% sodium deoxycholate) with a mixture of protease inhibitors (Calbiochem). Lysates were centrifuged at 16,600 × g to remove nucleic acids and cellular debris. Supernatants from 2 or more plates were pooled and precleared with protein G beads (Invitrogen) for 2 h at 4 °C. Protein lysates were separated into aliquots (150–225 μg of protein) and incubated with 3 μg of one of the following antibodies unless otherwise specified: EAAC1 (Alpha Diagnostics International, San Antonio, TX), EAAC1 (affinity purified, from Dr. J. D. Rothstein), Myc (BD Biosciences), NR1 (BD Pharmingen), NMDAR2C (1 μg; Invitrogen), rabbit IgG (Invitrogen), or mouse IgG (Invitrogen). After an overnight incubation at 4 °C, protein G beads (25 μl) were added and incubated at 4 °C for 2 h. Beads were isolated by centrifugation and subsequently washed four times with radioimmune precipitation assay buffer before the addition of 2× Laemmli sample buffer.Biotinylation Assays—Biotinylation of cell surface proteins was performed using hippocampal cultures or C6 glioma grown on 35- or 60-mm tissue culture dishes, as previously described (33Fournier K.M. Gonzaélez M.I. Robinson M.B. J. Biol. Chem. 2004; 279: 34505-34513Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Briefly, after rinsing cells with ice-cold phosphate-buffered saline supplemented with 0.1 mm calcium chloride and 1 mm magnesium chloride (phosphate-buffered saline (Ca2+/Mg2+)), plates were incubated with 1 mg/ml N-hydroxysulfosuccinimidobiotin (NHS-biotin, Pierce) in phosphate-buffered saline solution (Ca2+/Mg2+) for 30 min at 4 °C with slight agitation. After incubation, biotin was quenched using phosphate-buffered saline solution (Ca2+/Mg2+) containing 100 mm glycine for 30 min at 4 °C. Cells were lysed using radioimmune precipitation assay buffer containing 0.1% SDS and a mixture of protease inhibitors. Lysates were sonicated and centrifuged at 16,600 × g to remove nucleic acids and debris. Half of the lysate fraction was saved for analysis; the remaining lysate was agitated overnight at 4 °C with an equal volume of UltraLink immobilized monomeric avidin beads to isolate biotinylated proteins. The lysate-bead mixture was centrifuged, and the supernatant was retrieved (nonbiotinylated fraction) and saved for Western blot analysis. Beads were subsequently washed four times, and protein was eluted into SDS buffer with 2-mercaptoethanol (biotinylated fraction). The lysate, nonbiotinylated, and biotinylated fractions were all diluted in sample buffer such that the sum of the immunoreactivity in the biotinylated and the nonbiotinylated fractions should equal that observed in the lysate fraction if the yield from extraction is 100%.Western Blot Analyses—Western blot analyses were performed as previously described (38Guttmann R.P. Baker D.L. Seifert K.M. Cohen A.S. Coulter D.A. Lynch D.R. J. Neurochem. 2001; 78: 1083-1093Crossref PubMed Scopus (95) Google Scholar). Proteins were separated on an 8% SDS-PAGE gel. Antibodies directed toward these targets were utilized: EAAC1 (1:250; affinity-purified, from Dr. J. D. Rothstein), actin (1:10,000; Sigma), NR1 (1:1000; BD Pharmingen), NR1 (1:500; Chemicon International), NR2A (1:1500; Upstate Biotechnology, Charlottesville, VA), NR2B (1:500; Chemicon International MAB5220), NR2B (1:1000; Zymed Laboratories Inc., San Francisco, CA), GLT-1 (1:10,000; affinity purified, from Dr. J. D. Rothstein), GLAST (1:100; affinity purified, from J. D. Rothstein), PSD-95 (1:2000; BD Biosciences), GluR2 (1:1000; Chemicon International), Myc (1:1000; BD Biosciences), and NMDAR2C (1:2000; Invitrogen). Primary antibody incubation was followed by mouse monoclonal or rabbit polyclonal horseradish peroxidase secondary antibody (1:2500; Cell Signaling Technology, Danvers, MA). Immunoreactivity was visualized using enhanced chemiluminescence reagent (Pierce) and exposure on x-ray film. Bands were quantitated using densitometry normalized to actin (lysate) and then expressed as a percentage of control (no treatment) for each fraction. Glutamate transporters routinely form irreversible aggregates upon solubilization that are not dissociated with standard SDS-containing sample buffer (39Haugeto Ø. Ullensveng K. Levy L.M. Chaudhry F.A. Honore T. Neilsen M. Lehre K.P. Danbolt N.C. J. Biol. Chem. 1996; 271: 27715-27722Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Similar aggregates were observed in the present study. The amounts of monomeric and multimeric EAAC1 were quantitated and analyzed separately. Both species responded similarly, and therefore, data presented represent changes in the sum of the immunoreactivity of EAAC1 observed. Actin immunoreactivity in the biotinylated fraction was used to test for potential cell lysis. In all studies only a small percentage of actin immunoreactivity was detected in the biotinylated fraction (between 0 and 15%). In some cases data were represented as a percentage of transporter or receptor on the cell surface, calculated as (biotinylated/(biotinylated + nonbiotinylated)) × 100. When detecting more than one protein in a single experiment, nitrocellulose membranes were stripped (0.1 m glycine, pH 2.3 for 1 h) and then reprobed with the next antibody. Secondary antibodies were completely stripped before new antibody incubation. In instances where proteins of similar molecular weight were visualized (such as NR2A and NR2B), either primary antibodies requiring two different secondary antibodies were used, more than one gel was run with identical samples, or primary antibody was stripped off (confirmed by reincubating in secondary antibody and redeveloping).Statistical Analyses—Multiple comparisons were completed by analysis of variance followed by t test with Bonferroni corrections. Paired t test was used to directly compare values of two groups. When there were only two groups (vehicle and treatment) data were analyzed by one sample t test. For two-group comparisons with significantly different variances, data were analyzed using Mann-Whitney non-parametric tests. Statistical significance was set at a p < 0.05, computed using InStat (GraphPad Software, San Diego, CA).RESULTSInteraction of EAAC1 and NMDA Receptor Subunits—Because EAAC1 and ionotropic glutamate receptors may overlap in distribution (15Janssen W.G. Vissavajjhala P. Andrews G. Moran T. Hof P.R. Morrison J.H. Exp. Neurol. 2005; 191: S28-S44Crossref PubMed Scopus (50) Google Scholar, 17He Y. Janssen W.G.M. Rothstein J.D. Morrison J.H. J. Comp. Neurol. 2000; 418: 255-269Crossref PubMed Scopus (124) Google Scholar) and be regulated through similar mechanisms (Ref. 21Levenson J. Weeber E. Selcher J.C. Kategaya L.S. Sweatt J.D. Eskin A. Nat. Neurosci. 2002; 5: 155-161Crossref PubMed Scopus (124) Google Scholar; for a recent discussion, see Ref. 33Fournier K.M. Gonzaélez M.I. Robinson M.B. J. Biol. Chem. 2004; 279: 34505-34513Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), we examined potential interactions between EAAC1 and ionotropic glutamate receptors in primary hippocampal cultures. Using hippocampal culture lysates, a commercially available antibody specific to EAAC1 immunoprecipitated NMDA receptor subunits NR1, NR2A, and NR2B but not the AMPA receptor subunit GluR2 (Fig. 1A). Although a small amount of immunoreactivity of each NMDA receptor subunit was non-specifically detected with an IgG control, anti-EAAC1 always immunoprecipitated greater quantities of NMDA receptor subunit immunoreactivity. To verify the interaction between EAAC1 and NMDA receptors, immunoprecipitation was also performed using a different affinity purified EAAC1 antibody (from Dr. J. D. Rothstein). This antibody also immunoprecipitated NR1 (Fig. 1B). Because anti-EAAC1 immunoprecipitated NR1, NR2A, and NR2B, we aimed to identify the subpopulation of NMDA receptors with which EAAC1 may interact by immunoblotting for post-synaptic density 95 (PSD-95), a scaffolding protein of NMDA receptors in the postsynaptic density (40Sans N. Petralia R.S. Wang Y.X. Blahos 2nd, J. Hell J.W. Wenthold R.J. J. Neurosci. 2000; 20: 1260-1271Crossref PubMed Google Scholar). In these experiments one of the anti-EAAC1 antibodies (from Dr. J. D. Rothstein) immunoprecipitated PSD-95 (n = 2), whereas the other anti-EAAC1 antibody did not (n = 4). The former anti-EAAC1 antibody also immunoprecipitated more NR1 than the latter, although both antibodies immunoprecipitated comparable amounts of EAAC1 (n = 2).To determine whether this interaction can be observed in another system, we utilized a model in which NMDA receptor subunit composition could be manipulated. C6 glioma endogenously express EAAC1 (28Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar) but not NR1 (Fig. 1C, first lane). Small amounts of endogenous NR2A and NR2B were observed in C6 glioma (data not shown), but" @default.
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• W2083347740 date "2007-06-01" @default.
• W2083347740 modified "2023-09-30" @default.
• W2083347740 title "N-Methyl-d-aspartate Receptor-dependent Regulation of the Glutamate Transporter Excitatory Amino Acid Carrier 1" @default.
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• W2083347740 doi "https://doi.org/10.1074/jbc.m702278200" @default.
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