Key points are not available for this paper at this time.
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. 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. Bendahan A. Zafra F. Danbolt N.C. Aragón C. Giménez C. Kanner B.I. J. Biol. Chem. 1993; 268: 27313-27317Abstract Full Text PDF PubMed Google Scholar, 10Levy L.M. Lehre K.P. Walaas S.I. Storm-Mathisen J. Danbolt N.C. Eur. J. Neurosci. 1995; 7: 2036-2041Crossref PubMed Scopus (124) Google Scholar, 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) and highly differentially localized (12Torp R. Danbolt N.C. Babaie E. Bjørås M. Seeberg E. Storm-Mathisen J. Ottersen O.P. Eur. J. Neurosci. 1994; 6: 936-942Crossref PubMed Scopus (170) Google Scholar, 13Rothstein 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 (1449) Google Scholar, 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, 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 studies indicate that they modify glutamate receptor activation (16Barbour B. Keller B.U. Llano I. Marty A. Neuron. 1994; 12: 1331-1343Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 17Maki R. Robinson M.B. Dichter M.A. J. Neurosci. 1994; 14: 6754-6762Crossref PubMed Google Scholar, 18Mennerick S. Zorumsky C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (290) Google Scholar, 19Tong G. Jahr C.E. Neuron. 1994; 13: 1195-1203Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 20Takahashi M. Kovalchuck Y. Attwell D. J. Neurosci. 1995; 15: 5693-5702Crossref PubMed Google Scholar). 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)-3HGlutamic 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 The antibodies (rabbit (21Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (368) Google Scholar), were and purified against a purified glutamate transporter N.C. G. Kanner B.I. 1990; PubMed Scopus Google Scholar), were from the same purified as that previously described 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, 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 were by with GLAST from rat and antibodies from the using the The two anti-A522 antibodies from and labeling anti-A522 to antibodies from was with with in in Lowicryl HM20 at and as described using the same antibodies and 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). experiments between the labeling was by the of and PubMed Scopus Google Scholar), with as for the and for the one of the antibodies to of by the corresponding of in cells were in and with 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 (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 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, W. B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, B. Mol. Biol. 7: PubMed Scopus Google Scholar, G. Amara S.G. Biochem. PubMed Scopus Google Scholar). The was a from B. of The cells were from the and by for of both were by and The and were in of using a was not The was to cell membranes. of membranes from cells was in the same the membranes are referred to as membranes were prepared as described N.C. G. Kanner B.I. 1990; PubMed Scopus Google Scholar) by in of the and of the membranes after of of proteins in intact the (see were in buffer pH to a concentration of and in the or a cross-linker was suberate not to of or from a prepared in the was by pH to a concentration of the membranes were in buffer pH 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 with the buffer and on (see of for of were in of buffer and of membranes was with of ammonium sulfate and of solubilization buffer of buffer and pH with a or and the was with buffer and to of or The ammonium sulfate was with for experiments (see or the membranes were with of of of was with either anti-A522 or with and with buffer pH and The buffer the gel was were (see with were to of the of and of buffer pH were to each and or the were for reconstitution of transport (see and for The was from the by in buffer with and to The proteins were with or and The IgG that been in buffer and was by the of transporters were purified from rat by N.C. G. Kanner B.I. 1990; PubMed Scopus Google Scholar) and from the in buffer with a or was to the purified of the were with (see were in the the proteins on and the by with in the proteins were with high and and for transport (see of rat from were kept at and to using the at The of was using The were was in of the were to for at to that they the The were from to the was kept at was This and the equipment have been by inactivation of of molecular a of M. J. Sci. 1984; PubMed Scopus (46) Google Scholar, M. T. C. Biochem. Pharmacol. 34: PubMed Scopus Google Scholar, M. C. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). from the that the in the is and that is not required in However, to the of the the of and to and the of were The were in buffer (50 pH and and The were for using the The were either used for of as described M. T. C. Biochem. Pharmacol. 34: PubMed Scopus Google Scholar) or with of solubilization buffer pH on and as The were in of and to reconstitution (see of in was as described 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, N.C. G. Kanner B.I. 1990; PubMed Scopus Google Scholar). with glutamate transporters and either or was with of a on and to on with the of was as described N.C. G. Kanner B.I. 1990; PubMed Scopus Google Scholar). the uptake was by of with amino acid and The was by and The were with the R. A. Biochem. J. PubMed Scopus Google and (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google Scholar, Nature. PubMed Scopus Google Scholar) was with of or The electrophoresis with kept at electrophoresis the proteins were either N.C. G. Kanner B.I. 1990; PubMed Scopus Google Scholar) or 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, H. T. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The were to (15Lehre K.P. Levy L.M. Ottersen O.P. Storm-Mathisen J. Danbolt N.C. J. Neurosci. 1995; 15: 1835-1853Crossref PubMed Google of were by the of R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) using as IgG was at using IgG as 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. Bendahan A. Zafra F. Danbolt N.C. Aragón C. Giménez C. Kanner B.I. J. Biol. Chem. 1993; 268: 27313-27317Abstract Full Text PDF PubMed Google Scholar, 10Levy L.M. Lehre K.P. Walaas S.I. Storm-Mathisen J. Danbolt N.C. Eur. J. Neurosci. 1995; 7: 2036-2041Crossref PubMed Scopus (124) Google Scholar, 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) and highly differentially localized (12Torp R. Danbolt N.C. Babaie E. Bjørås M. Seeberg E. Storm-Mathisen J. Ottersen O.P. Eur. J. Neurosci. 1994; 6: 936-942Crossref PubMed Scopus (170) Google Scholar, 13Rothstein 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 (1449) Google Scholar, 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, 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 studies indicate that they modify glutamate receptor activation (16Barbour B. Keller B.U. Llano I. Marty A. Neuron. 1994; 12: 1331-1343Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 17Maki R. Robinson M.B. Dichter M.A. J. Neurosci. 1994; 14: 6754-6762Crossref PubMed Google Scholar, 18Mennerick S. Zorumsky C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (290) Google Scholar, 19Tong G. Jahr C.E. Neuron. 1994; 13: 1195-1203Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 20Takahashi M. Kovalchuck Y. Attwell D. J. Neurosci. 1995; 15: 5693-5702Crossref PubMed Google Scholar). 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
Haugeto et al. (Fri,) studied this question.
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