Brain Glutamate Transporter Proteins Form Homomultimers
1996; Elsevier BV; Volume: 271; Issue: 44 Linguagem: Inglês
10.1074/jbc.271.44.27715
ISSN1083-351X
AutoresØyvind Haugeto, Kyrre Ullensvang, Line M. Levy, Farrukh A. Chaudhry, Tage Honoré, Mogens Brøndsted Nielsen, Knut P. Lehre, Niels C. Danbolt,
Tópico(s)Ion channel regulation and function
ResumoRemoval of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes. Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes. INTRODUCTIONThe majority of excitatory signals in the mammalian central nervous system may be transmitted by glutamate (1Fonnum F. J. Neurochem. 1984; 42: 1-11Crossref PubMed Scopus (1670) Google Scholar). The extracellular glutamate concentration has to be kept low, both to secure a high signal-to-noise (background) ratio and because excessive glutamate receptor activation can lead to neuronal damage (2Olney J.W. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 47-71Crossref PubMed Scopus (378) Google Scholar). This is achieved by the action of sodium-dependent glutamate transporters located in the plasma membranes of both glial cells and neurons (3Danbolt N.C. Prog. Neurobiol. (New York). 1994; 44: 377-396Crossref PubMed Scopus (213) Google Scholar). Several glutamate transporters have been cloned: GLAST 1The abbreviations used are: GLASTrat glutamate aspartate transporter (4Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1092) Google Scholar)CHAPS3-[(3-cholamido-propyl)dimethylammonio]- 1-propanesulfonateDTNB5,5′-dithio-bis(2-nitrobenzoic acid)DTTdithiothreitolEAACan EAAC1-type transporterEAAC1rabbit excitatory amino acid carrier (7Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1192) Google Scholar)GLTa GLT-1-type transporterGLT-1rat glutamate transporter (5Pines G. Danbolt N.C. Bjørås M. Zhang Y. Bendahan A. Eide L. Koepsell H. Seeberg E. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1131) Google Scholar6Kanner B.I. FEBS Lett. 1993; 325: 95-99Crossref PubMed Scopus (123) Google Scholar)GLYT1rat glycine transporter (33Smith K.E. Borden L.A. Hartig P.R. Branchek T. Weinshank R.L. Neuron. 1992; 8: 927-935Abstract Full Text PDF PubMed Scopus (379) Google Scholar)NaPisodium phosphate buffer with pH 7.4PMSFphenylmethanesulfonyl fluoriderEAAC1rat excitatory amino acid carrier (36Bjørås M. Gjesdal O. Erickson J.D. Torp R. Levy L.M. Ottersen O.P. Degree M. Storm-Mathisen J. Seeberg E. Danbolt N.C. Mol. Brain Res. 1996; 36: 163-168Crossref PubMed Scopus (58) Google Scholar)PAGEpolyacrylamide gel electrophoresisTEMEDN,N,N′,N′-tetramethylethylenediamine. (4Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1092) Google Scholar), GLT-1 (5Pines G. Danbolt N.C. Bjørås M. Zhang Y. Bendahan A. Eide L. Koepsell H. Seeberg E. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1131) Google Scholar, 6Kanner B.I. FEBS Lett. 1993; 325: 95-99Crossref PubMed Scopus (123) Google Scholar), EAAC1 (7Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1192) Google Scholar), and EAAT4 (8Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1006) Google Scholar). The transporters are regulated (9Casado M. 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Thus, the functions of these carriers may be more refined than simple removal of excitatory amino acids.It is legitimate to ask whether the glutamate transporters might form oligomeric complexes. Several glutamate transporter subtypes exist (see above). These proteins have been reported to aggregate (21Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (368) Google Scholar, 22Levy L.M. Lehre K.P. Rolstad B. Danbolt N.C. FEBS Lett. 1993; 317: 79-84Crossref PubMed Scopus (112) Google Scholar, 23Rothstein J.D. Van Kammen M. Levey A.I. Martin L.J. Kuncl R.W. Ann. Neurol. 1995; 38: 73-84Crossref PubMed Scopus (1189) Google Scholar). GLAST and GLT have been observed in the same cells (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google Scholar). Recent reports conclude that some other co-transporters may exist in vivo as oligomers (24Béliveau R. Demeule M. Ibnoul-Khatib H. Bergeron M. Beauregard G. Potier M. Biochem. J. 1988; 252: 807-813Crossref PubMed Scopus (43) Google Scholar, 25Hebert D.N. Carruthers A. J. Biol. Chem. 1992; 267: 23829-23838Abstract Full Text PDF PubMed Google Scholar, 26Berger S.P. Farrell K. Conant D. Kempner E.S. Paul S.M. Mol. Pharmacol. 1994; 46: 726-731PubMed Google Scholar, 27Milner H.E. Béliveau R. Jarvis S.M. Biochim. Biophys. Acta. 1994; 1190: 185-187Crossref PubMed Scopus (46) Google Scholar, 28Wang Y. Tate S.S. FEBS Lett. 1995; 368: 389-392Crossref PubMed Scopus (61) Google Scholar. The glutamate transporters behave like a combination of carriers and chloride channels (18Mennerick S. Zorumsky C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (290) Google Scholar, 29Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (451) Google Scholar). Several neurotransmitter receptors, including ionotropic glutamate receptors, operate as hetero-oligomeric complexes (30McBain C.J. Mayer M.L. Physiol. Rev. 1994; 74: 723-760Crossref PubMed Scopus (0) Google Scholar, 31Bettler B. Mulle C. Neuropharmacology. 1995; 34: 123-139Crossref PubMed Scopus (419) Google Scholar).Here we show by double labeling post-embedding electron microscopic immunocytochemistry that the two glial glutamate transporters GLT and GLAST are expressed in the same cell membranes. Furthermore, we demonstrate that GLT and GLAST, as well as the neuronal glutamate transporter EAAC, form oligomeric complexes but that GLT and GLAST do not complex with each other. Evidence suggests that oligomeric structure is required for transport activity.EXPERIMENTAL PROCEDURESMaterials—Sodium dodecyl sulfate (SDS) of high purity (.99% C12 alkyl sulfate) and bis(sulfosuccinimidyl) suberate were from Pierce. Nitrocellulose sheets (0.22-mm pores, 100% nitrocellulose) and electrophoresis equipment were from Hoefer Scientific Instruments (San Francisco, CA). N,N9-Methylenebisacrylamide, acrylamide, ammonium persulfate, TEMED, and alkaline phosphatase substrates (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) were from Promega (Madison, WI). Alkaline phosphatase conjugated to mouse monoclonal anti-rabbit IgG (A-2556, clone RG-96) was obtained from Sigma and used at 1:10,000. Glutaraldehyde, EM grade, was from TAAB (Reading, UK). Lowicryl HM20 was from (Lowi, Switzerland). (S)-[3H]Glutamic acid (50 Ci/mmol), molecular mass markers for SDSpolyacrylamide gel electrophoresis (SDS-PAGE), and colloidal gold-labeled second antibodies (GAR15, GAR30) were from Amersham (Buckinghamshire, UK). Cholic acid was purified with activated charcoal and by recrystallization from 70% ethanol. Wheat germ agglutinin was immobilized to agarose as described previously (21,). All other reagents were either obtained from Sigma or from Fluka (Buchs, Switzerland).Production of Antibodies—Anti-peptide antibodies against three glutamate transporters (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google Scholar) and one glycine transporter (32Zafra F. Aragón C. Olivares L. Danbolt N.C. Giménez C. Storm-Mathisen J. J. Neurosci. 1995; 15: 3952-3969Crossref PubMed Google Scholar) were prepared as described using synthetic peptides as antigens. The peptides representing parts of GLAST (rat EAAT1), GLT-1 (rat EAAT2), EAAC1 (rabbit EAAT3), and GLYT1 are referred to by capital letters A, B, C, and G, respectively, followed by numbers indicating the corresponding amino acid residues in the sequences (given in parentheses): A522-541 (PYQLIAQDNEPEKPVADSET), B12-26 (KQVEVRMHDSHLSSE), B493-508 (YHLSKSELDTIDSQHR), C510-524 (VDKSDTISFTQTSQF), and G623-638 (IVGSNGSSRLQDSRI) (4Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1092) Google Scholar, 5Pines G. Danbolt N.C. Bjørås M. Zhang Y. Bendahan A. Eide L. Koepsell H. Seeberg E. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1131) Google Scholar, 7Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1192) Google Scholar, 33Smith K.E. Borden L.A. Hartig P.R. Branchek T. Weinshank R.L. Neuron. 1992; 8: 927-935Abstract Full Text PDF PubMed Scopus (379) Google Scholar). The corresponding anti-peptide antibodies are referred to as anti-A522 (rabbit 68488), anti-B12 (rabbit 68518), anti-B493 (rabbit 84946), anti-C510 (rabbit 69738), or anti-G623 (rabbit 80748). The anti-73-kDa antibodies (rabbit 85302) (21Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (368) Google Scholar), which were raised and affinity purified against a purified glutamate transporter (34Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar), were from the same purified batch as that previously described (14Chaudhry F.A. Lehre K.P. van Lookeren-Campagne M. Ottersen O.P. Danbolt N.C. Storm-Mathisen J. Neuron. 1995; 15: 711-720Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 21Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (368) Google Scholar, 22Levy L.M. Lehre K.P. Rolstad B. Danbolt N.C. FEBS Lett. 1993; 317: 79-84Crossref PubMed Scopus (112) Google Scholar). The anti-A522 antibodies from rabbit 20604 were produced by immunizing with whole GLAST protein isolated from rat cerebellum and affinity purifying antibodies from the crude serum using the A522 peptide. The two anti-A522 antibodies from rabbit 68488 and 20604 gave identical immunoblot labeling patterns. Unless stated otherwise, anti-A522 refers to antibodies from rabbit 20604.Electron Microscopic ImmunocytochemistryTissue was perfusion-fixed with 2.5% glutaraldehyde, 1% formaldehyde, freeze-substituted with 0.5% uranyl acetate in methanol, embedded in Lowicryl HM20 at low temperature, and further processed as described before using the same antibodies and concentrations (14Chaudhry F.A. Lehre K.P. van Lookeren-Campagne M. Ottersen O.P. Danbolt N.C. Storm-Mathisen J. Neuron. 1995; 15: 711-720Abstract Full Text PDF PubMed Scopus (693) Google Scholar). Immunoblot experiments showed no cross-reactivity between the antibodies. Double immunogold labeling was performed by the method of Wang and Larsson (35Wang B.L. Larsson L.I. Histochemistry. 1985; 83: 47-56Crossref PubMed Scopus (208) Google Scholar), with GAR30 (30-nm gold particles) as reporter for the first primary antibody and GAR15 (15 nm) for the second. Omitting one of the primary antibodies leads to absence of label by the corresponding reporter particle type.Expression of Glutamate Transporters in HeLa CellsHeLa cells were maintained in monolayer culture and transfected with cDNAs encoding GLT (5Pines G. Danbolt N.C. Bjørås M. Zhang Y. Bendahan A. Eide L. Koepsell H. Seeberg E. Storm-Mathisen J. Seeberg E. Kanner B.I. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1131) Google Scholar) or rEAAC1 (36Bjørås M. Gjesdal O. Erickson J.D. Torp R. Levy L.M. Ottersen O.P. Degree M. Storm-Mathisen J. Seeberg E. Danbolt N.C. Mol. Brain Res. 1996; 36: 163-168Crossref PubMed Scopus (58) Google Scholar) using the vaccinia virus T7 expression system (36Bjørås M. Gjesdal O. Erickson J.D. Torp R. Levy L.M. Ottersen O.P. Degree M. Storm-Mathisen J. Seeberg E. Danbolt N.C. Mol. Brain Res. 1996; 36: 163-168Crossref PubMed Scopus (58) Google Scholar, 37Fuerst T.R. Niles E.G. Studier W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1864) Google Scholar, 38Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (331) Google Scholar, 39Blakely R.D. Clark J.A. Rudnick G. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar). The virus was a generous gift from B. Moss (National Institutes of Health). The transfected HeLa cells were detached from the flasks and collected by centrifugation (1000 rpm, 5 min).Tissue Preparation for BiochemistryWistar rats (180-250 g) of both sexes (Møllegaard Hansen, Denmark) were killed by stunning and decapitation. The forebrain and cerebellum were dissected, homogenized immediately in 10-20 volumes of ice-cold hypotone solution (5 mM EDTA, 1 mM PMSF) using a Dounce glass-glass homogenizer. DTT (5 mM) was added when not stated otherwise. The homogenate was centrifuged (18,000 rpm, 39,000 × g, 4°C, 15 min) to sediment cell membranes. Preparation of membranes from transfected HeLa cells was done in the same way. Below, the sedimented membranes are referred to as "the membrane pellet." When stated, membranes were prepared as described (34Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar) by homogenizing in 0.32 M mannitol, isolation of the crude synaptosomal fraction, and sedimentation of the membranes after osmotic shock.Cross-linking of Membrane ProteinsFor cross-linking of proteins in intact membranes, the membrane pellets (see under "Tissue Preparation" above) were resuspended in buffer (150 mM NaCl, 100 mM Na-HEPES, pH 7.5, 5 mM EDTA, 1 mM PMSF, 5 mM DTT) to a final protein concentration of about 0.5 mg/ml and divided in aliquots. To the membrane suspensions, protein solutions, or liposomes a cross-linker was added (bis(sulfosuccinimidyl) suberate when not stated otherwise) to final concentrations of 0.3, 1, 3, 10, or 30 mM from a freshly prepared 100 mM stock solution in 20 mM HCl. After incubation (12 min, room temperature, end-over-end mixing), the reaction was terminated by adding 2 M Tris-HCl, pH 9, to a final concentration of 200 mM. Then the membranes were solubilized in SDS-sample buffer (70 mM SDS, 62.5 mM Tris-HCl, pH 6.8, 0.3 M sucrose, 10 µg/ml bromphenol blue), gel-filtered (11Trotti D. Volterra A. Lehre K.P. Rossi D. Gjesdal O. Racagni G. Danbolt N.C. J. Biol. Chem. 1995; 270: 9890-9895Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) on Sephadex G-50 fine spin columns equilibrated with the above SDS-sample buffer (containing 5 mM DTT), and run on SDS-PAGE (see below).Solubilization of Membranes for Cross-linking of Solubilized ProteinsThe above membrane pellets were resuspended in equal volumes of buffer (150 mM NaCl, 100 mM Na-HEPES) and divided into aliquots. One volume of resuspended membranes was mixed with 0.35 volumes of saturated ammonium sulfate and 2 volumes of solubilization buffer consisting of buffer (5 mM EDTA, 1 mM PMSF, 5 mM DTT, and 100 mM Na-HEPES, pH 7.5) with a detergent (33 mM CHAPS, 3.3 µl/ml Triton X-100, or 50 mM cholate). After incubation (10 min, 0°C) and centrifugation (39,000 × g, 18,000 rpm, 4°C, 20 min), the supernatant was diluted 1 + 4 with buffer and detergent to give final detergent concentrations of 20 mM CHAPS, 1 µl/ml Triton X-100, or 30 mM cholate. The saturated ammonium sulfate was replaced with 5 M NaCl for cross-linking experiments (see below) or omitted when the membranes were solubilized with 1% SDS.Immunoabsorption of Glutamate TransportersAliquots of 100 µl of protein A-Sepharose Fast Flow was mixed with either anti-B12, anti-B493, anti-A522 or preimmune IgG, incubated (1 h, room temperature) with 20% newborn calf serum, and equilibrated with buffer (500 mM NaCl, 5 mM EDTA, 1 mM PMSF, 10 mM NaPi, pH 7.4, 0.1% Tween 20 and 0.05% NaN3). The buffer above the settled gel surface was removed. Membranes were solubilized (see above) with cholate. Reducing agents were omitted to avoid reduction of the antibodies. 200 µl of extract and 80 µl of buffer (500 mM NaCl, 1.25% cholate, 5 mM EDTA, 1 mM PMSF, 10 mM NaPi pH 7.4) were added to each protein A-Sepharose tube and incubated (4°C, end-over-end, 30 or 60 min). Then the supernatants were collected for reconstitution of transport activity (see below) and for SDS-PAGE. The precipitated protein was released from the Sepharose beads by boiling in SDS-sample buffer with 5% mercaptoethanol and subjected to SDS-PAGE. The proteins were immunoblotted with biotinylated (not shown) or regular non-biotinylated (Fig. 4) anti-GLT and anti-GLAST antibodies. The rabbit IgG that had been boiled in SDS-sample buffer containing mercaptoethanol and blotted, was poorly detected by the secondary antibody used.Partial Purification of Glutamate TransportersGlutamate transporters were partially purified from rat forebrain by lectin affinity chromatography (34Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar) and eluted from the columns in buffer with a detergent (20 mM CHAPS, 50 mMβ-octylglucoside, or 15 mM Zwittergent 3-12). 10 mM DTT was added to the purified fractions. Some aliquots of the fractions were mixed with cross-linkers (see below). Other aliquots were incubated (15 min, 0°C) in the elution buffer, the proteins collected on DEAE-cellulose columns, and the detergent removed by washing with 20 mM CHAPS in 20 mM NaPi. Then the proteins were released with high salt (500 mM NaCl, 20 mM CHAPS, and 50 mM NaPi), reconstituted, and assayed for transport activity (see below).Radiation Inactivation AnalysisSamples of rat cerebral cortex (0.2-0.35 g) from male Wistar rats (180 g) were kept at −20°C and exposed to high-energy electrons using the 10 MeV linear accelerator at Risø, Denmark. The dose of radiation was determined using calibrated thermodosimeters (water). The samples were frozen (−10°C) during radiation which was delivered in runs of 0.5-2 Mrad. Between runs the samples were cooled to −15°C for at least 2 min to ensure that they remained completely frozen during the entire irradiation process. The total doses were from 0 to 20 Mrad. After radiation, the tissue was kept at −80°C until it was used. This irradiation procedure and the equipment have been calibrated by radiation inactivation of seven enzymes of known molecular masses (67-148 kDa) which gave a calibration constant of 730,000 Da × Mrad (40Nielsen M. Klimek V. Hyttel J. Life Sci. 1984; 35: 325-332Crossref PubMed Scopus (46) Google Scholar, 41Nielsen M. Honoré T. Braestrup C. Biochem. Pharmacol. 1985; 34: 3633-3642Crossref PubMed Scopus (64) Google Scholar, 42Nielsen M. Braestrup C. J. Biol. Chem. 1988; 263: 11900-11906Abstract Full Text PDF PubMed Google Scholar). Experience from the last 10 years shows that the interexperimental variation in the radiation effect is negligible and that extensive calibration is not required in every experiment. However, to verify the validity of the calibration, the binding of [3H]flunitrazepam and [3H]RO 15-1788 to benzodiazepine receptors and the activity of pyruvate kinase were determined. The irradiated samples were homogenized in buffer (50 mM Tris acetate, pH 7.4, 5 mM EDTA, and 1 mM PMSF) and centrifuged (39,000 × g, 18,000 rpm, 20 min, 4°C). The supernatants were assayed for pyruvate kinase activity using the Boehringer Mannheim test kit 126047. The pellets were either used for determination of benzodiazepine binding as described (41Nielsen M. Honoré T. Braestrup C. Biochem. Pharmacol. 1985; 34: 3633-3642Crossref PubMed Scopus (64) Google Scholar) or mixed with 12 volumes of solubilization buffer (32 mM cholate, 500 mM NaCl, 5 mM EDTA, 100 mM NaPi, pH 7.4), incubated (10 min) on ice, and centrifuged as above. The supernatants were immediately frozen (−80°C) in portions of 300 µl and thawed immediately prior to reconstitution (see below).Reconstitution of Glutamate Transporters in LiposomesThis was done as described (11Trotti D. Volterra A. Lehre K.P. Rossi D. Gjesdal O. Racagni G. Danbolt N.C. J. Biol. Chem. 1995; 270: 9890-9895Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 34Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar). Briefly, 100-µl sample with glutamate transporters and either 20 mM CHAPS, 30 mM cholate, or 1 µl/ml Triton X-100 was mixed with 150 µl of a phospholipid/cholate/salt mixture, incubated on ice, and gel-filtered to remove detergent on spin columns equilibrated with the desired internal medium.Determination of Glutamate Transport ActivityThis was done exactly as described (34Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar). Briefly, the uptake reaction was started by diluting 20 µl of proteoliposomes into saline with tritiated amino acid and valinomycin. The reaction was terminated by dilution and filtration. The kinetic data were analyzed with the direct linear plot method (43Eisenthal R. Cornish-Bowden A. Biochem. J. 1974; 139: 715-720Crossref PubMed Scopus (1236) Google Scholar).Electrophoresis and BlottingSDS-PAGE (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google Scholar, 44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar) was done with separating gels consisting of 5 or 10% acrylamide. The electrophoresis trays had heat exchangers with cooling water kept at 0-5°C. After electrophoresis the proteins were either silver-stained (34Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar) or electroblotted onto nitrocellulose membranes (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google Scholar, 45Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44708) Google Scholar). The blots were usually allowed to dry before immunostaining (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google Scholar).Protein DeterminationConcentrations of membrane protein were determined by the method of Lowry et al. (46Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. Purified IgG was quantified spectrophotometrically at 280 nm using bovine IgG as standard. INTRODUCTIONThe majority of excitatory signals in the mammalian central nervous system may be transmitted by glutamate (1Fonnum F. J. Neurochem. 1984; 42: 1-11Crossref PubMed Scopus (1670) Google Scholar). The extracellular glutamate concentration has to be kept low, both to secure a high signal-to-noise (background) ratio and because excessive glutamate receptor activation can lead to neuronal damage (2Olney J.W. Annu. Rev. Pharmacol. 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The glutamate transporters behave like a combination of carriers and chloride channels (18Mennerick S. Zorumsky C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (290) Google Scholar, 29Wadiche J.I. Amara S.G. Kavanaugh M.P. Neuron. 1995; 15: 721-728Abstract Full Text PDF PubMed Scopus (451) Google Scholar). Several neurotransmitter receptors, including ionotropic glutamate receptors, operate as hetero-oligomeric complexes (30McBain C.J. Mayer M.L. Physiol. Rev. 1994; 74: 723-760Crossref PubMed Scopus (0) Google Scholar, 31Bettler B. Mulle C. Neuropharmacology. 1995; 34: 123-139Crossref PubMed Scopus (419) Google Scholar).Here we show by double labeling post-embedding electron microscopic immunocytochemistry that the two glial glutamate transporters GLT and GLAST are expressed in the same cell membranes. Furthermore, we demonstrate that GLT and GLAST, as well as the neuronal glutamate transporter EAAC, form oligomeric complexes but that GLT and GLAST do not complex with each other. Evidence suggests that oligomeric structure is required for transport activity.
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