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Inherited Defects of Sodium-dependent Glutamate Transport Mediated by Glutamate/Aspartate Transporter in Canine Red Cells Due to a Decreased Level of Transporter Protein Expression

2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês

10.1074/jbc.275.9.6620

1083-351X

Kota Sato, Mutsumi Inaba, Yuki Suwa, Aya Matsuu, Yoshiaki Hikasa, Kenichiro Ono, Katsumoto KAGOTA,

Neurogenesis and neuroplasticity mechanisms

Canine red cells have a high affinity Na+/K+-dependent glutamate transporter. We herein demonstrate that this transport is mediated by the canine homologue of glutamate/aspartate transporter (GLAST), one of the glutamate transporter subtypes abundant in the central nervous system. We also demonstrate that GLAST is the most ubiquitous glutamate transporter among the transporter subtypes that have been cloned to date. The GLAST protein content was extremely reduced in variant red cells, low glutamate transport (LGlut) red cells characterized by an inherited remarkable decrease in glutamate transport activity. All LGluT dogs carried a missense mutation of Gly492 to Ser (G492S) in either the heterozygous or homozygous state. The GLAST protein with G492S mutation was fully functional in glutamate transport in Xenopus oocytes. However, G492S GLAST exhibited a marked decrease in activity after the addition of cycloheximide, while the wild type showed no significant change, indicating that G492S GLAST was unstable compared with the wild-type transporter. Moreover, LGluT dogs, but not normal dogs, heterozygous for the G492S mutation showed a selective decrease in the accumulation of GLAST mRNA from the normal allele. Based on these findings, we conclude that a complicated heterologous combination of G492S mutation and some transcriptional defect contributes to the pathogenesis of the LGluT red cell phenotype. Canine red cells have a high affinity Na+/K+-dependent glutamate transporter. We herein demonstrate that this transport is mediated by the canine homologue of glutamate/aspartate transporter (GLAST), one of the glutamate transporter subtypes abundant in the central nervous system. We also demonstrate that GLAST is the most ubiquitous glutamate transporter among the transporter subtypes that have been cloned to date. The GLAST protein content was extremely reduced in variant red cells, low glutamate transport (LGlut) red cells characterized by an inherited remarkable decrease in glutamate transport activity. All LGluT dogs carried a missense mutation of Gly492 to Ser (G492S) in either the heterozygous or homozygous state. The GLAST protein with G492S mutation was fully functional in glutamate transport in Xenopus oocytes. However, G492S GLAST exhibited a marked decrease in activity after the addition of cycloheximide, while the wild type showed no significant change, indicating that G492S GLAST was unstable compared with the wild-type transporter. Moreover, LGluT dogs, but not normal dogs, heterozygous for the G492S mutation showed a selective decrease in the accumulation of GLAST mRNA from the normal allele. Based on these findings, we conclude that a complicated heterologous combination of G492S mutation and some transcriptional defect contributes to the pathogenesis of the LGluT red cell phenotype. glutamate/aspartate transporter glutamate transporter-1 excitatory amino acid carrier 1 excitatory amino acid transporter 4 and 5, respectively polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid low glutamate transport normal glutamate transport polymerase chain reaction reverse transcriptase-PCR nucleotides High affinity Na+-dependent glutamate transporters play important physiological roles in various mammalian tissues and cells. Several distinct glutamate transporters, GLAST,1 GLT-1, EAAC1, EAAT4, and EAAT5 have been cloned, and their electrophysiological and pharmacological properties have been characterized (1.Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1097) Google Scholar, 2.Pines 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 (1136) Google Scholar, 3.Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1197) Google Scholar, 4.Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1013) Google Scholar, 5.Arriza J.L. Eliasof S. Kavanaugh M.P. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4155-4160Crossref PubMed Scopus (801) Google Scholar). In the central nervous system, these transporters are highly differentially localized and participate in glutamate uptake into glial or neuronal cells to terminate excitatory neurotransmission (6.Nicholls D. Attwell D. Trends Pharmacol. Sci. 1990; 11: 462-468Abstract Full Text PDF PubMed Scopus (973) Google Scholar). In particular, glial transporters GLAST and GLT-1 play critical roles to maintain the extracellular glutamate concentration at the submicromolar level, thereby preventing accumulation of glutamate in the synaptic cleft, which causes overstimulation of the receptors and neurodegeneration. Dysfunction of glutamate transporters has been considered to be involved in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (7.Lin C.L. Bristol L.A. Jin L. Dykes-Hoberg M. Crawford T. Clawson L. Rothstein J.D. Neuron. 1998; 20: 589-602Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 8.Trotti D. Danbolt N.C. Volterra A. Trends Pharmacol. Sci. 1998; 19: 328-334Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar), Alzheimer's disease, trauma, and ischemia (8.Trotti D. Danbolt N.C. Volterra A. Trends Pharmacol. Sci. 1998; 19: 328-334Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). In peripheral tissues, glutamate transporters are believed to have pivotal functions in epithelial transport and absorption of acidic amino acids (9.Schneider E.G. Hammerman M.R. Sactor B. J. Biol. Chem. 1980; 255: 7650-7656Abstract Full Text PDF PubMed Google Scholar, 10.Rajendran V.M. Harig J.M. Adams M.B. Ramaswamy K. Am. J. Physiol. 1987; 252: G33-G39PubMed Google Scholar) and in modulation of glutathione synthesis (11.Deneke S.M. Steiger V. Fanburg B.L. J. Appl. Physiol. 1987; 63: 1966-1971Crossref PubMed Scopus (45) Google Scholar). EAAC1 is presumed to be a transporter in the epithelia of the intestine and kidney, because its transcripts were identified in those peripheral tissues as well as in neurons (3.Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1197) Google Scholar). However, physiological and pathological functions of other transporter isoforms in peripheral tissues have not been well characterized. We have been interested in the structure, function, and regulation of expression of the red cell glutamate transporter in dogs. Canine red cells possess a high affinity Na+- and K+-dependent l-glutamate andl-aspartate transport system (12.Inaba M. Maede Y. J. Biol. Chem. 1984; 259: 312-317Abstract Full Text PDF PubMed Google Scholar, 13.Sato K. Inaba M. Maede Y. Biochim. Biophys. Acta. 1994; 1195: 211-217Crossref PubMed Scopus (9) Google Scholar), despite the fact that most mammalian red cells are impermeable to these acidic amino acids (14.Young J.D. Ellory J.C. Ellory J.C. Lew V.L. Membrane Transport in Red Cells. Academic Press, London1977: 301-325Google Scholar). Dogs usually have red cells with low K+ and high Na+ concentrations (LK red cells) because they lose red cell Na,K-ATPase during reticulocyte maturation (15.Maede Y. Inaba M. J. Biol. Chem. 1985; 260: 3337-3343Abstract Full Text PDF PubMed Google Scholar, 16.Inaba M. Maede Y. J. Biol. Chem. 1986; 261: 16099-16105Abstract Full Text PDF PubMed Google Scholar). However, some Japanese Shiba and mongrel dogs have HK red cells with high Na,K-ATPase activity, resulting in high K+ and low Na+ concentrations, and this HK phenotype is inherited in an autosomal recessive manner (17.Maede Y. Inaba M. Taniguchi N. Blood. 1983; 61: 493-499Crossref PubMed Google Scholar). HK red cells show accelerated Na+/K+-dependent glutamate/aspartate uptake due to an increased concentration gradient of Na+ and K+ across the plasma membrane, leading to marked accumulations of intracellular glutamate, aspartate, and glutamine (12.Inaba M. Maede Y. J. Biol. Chem. 1984; 259: 312-317Abstract Full Text PDF PubMed Google Scholar, 17.Maede Y. Inaba M. Taniguchi N. Blood. 1983; 61: 493-499Crossref PubMed Google Scholar). The increased concentration of glutamate further results in an elevated level of reduced glutathione and affects the redox state and protection against oxidative stress of the red cell (18.Maede Y. Kuwabara M. Sasaki A. Inaba M. Hiraoka W. Blood. 1989; 73: 312-317Crossref PubMed Google Scholar). Interestingly, these breeds also include dogs characterized by reduced (19.Fujise H. Hamada Y. Mori M. Ochiai H. Biochim. Biophys. Acta. 1995; 1239: 22-26Crossref PubMed Scopus (17) Google Scholar) and nondetectable 2K. Sato, M. Inaba, and Y. Maede, unpublished observation. 2K. Sato, M. Inaba, and Y. Maede, unpublished observation. red cell glutamate transport, generating variant HK cells without accumulation of glutathione. Their red cells were readily accessible to oxidants such as acetylphenyl hydrazine and generated many more Heinz bodies than normal HK cells or even LK red cells did. 3K. Sato, M. Inaba, and K. Kagota, unpublished observation. 3K. Sato, M. Inaba, and K. Kagota, unpublished observation. Such a hereditary defect of the glutamate transporter in mammals has never been described so far as we know, although several pathological studies on mice lacking glutamate transporters due to gene disruption has been reported (20.Tanaka 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 (1474) Google Scholar, 21.Peghini P. Janzen J. Stoffel W. EMBO J. 1997; 16: 3822-3832Crossref PubMed Scopus (274) Google Scholar, 22.Watase K. Hashimoto K. Kano M. Yamada K. Watanabe M. Inoue Y. Okuyama S. Sakagawa T. Ogawa S. Kawashima N. Hori S. Takimoto M. Wada K. Tanaka K. Eur. J. Neurosci. 1998; 10: 976-988Crossref PubMed Scopus (356) Google Scholar). The observations in dogs suggested that the functions of the glutamate transporters contributed to protect cellular contents from oxidative damage in peripheral tissues. Defining the molecular basis that underlies the transport deficiency in canine red cells may facilitate our understanding of the regulatory mechanisms for expression of, and the physiological and pathological roles for, the glutamate transporter in various tissues, including the brain. We have postulated that, based on observations of its kinetic and pharmacologic properties, the canine red cell glutamate transporter is EAAC1 (13.Sato K. Inaba M. Maede Y. Biochim. Biophys. Acta. 1994; 1195: 211-217Crossref PubMed Scopus (9) Google Scholar). The purpose of the present study is to precisely define the glutamate transporter in canine red cells, thereby clarifying the underlying cause for the hereditary deficiency of the transport. The dogs used in this study were from a family of Japanese mongrel dogs that were a mixed breed of Japanese Shiba. Some pure Shiba and Beagle dogs were also used. These dogs were clinically healthy, and hematological parameters of their red cells were within reference ranges except that HK red cells had a mean corpuscular volume slightly larger than that of LK red cells as demonstrated before (17.Maede Y. Inaba M. Taniguchi N. Blood. 1983; 61: 493-499Crossref PubMed Google Scholar). Methods for isolation of total RNA, poly(A)+ RNA, and genomic DNA, reverse transcription, PCR, and cloning of PCR products were described previously (23.Inaba M. Yawata A. Koshino I. Sato K. Takeuchi M. Takakuwa Y. Manno S. Yawata Y. Kanzaki A. Sakai J. Ban A. Ono K. Maede Y. J. Clin. Invest. 1996; 97: 1804-1817Crossref PubMed Scopus (163) Google Scholar). RNAs were treated with DNase I. DNA sequencing was carried out using a Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) on an automated DNA sequencer ALF express (Amersham Pharmacia Biotech, Uppsala, Sweden) or using a BigDye primer or terminator cycle sequencing kit (Perkin-Elmer Applied Biosystems, Foster City, CA) on a 377A DNA sequencer. Total RNA from various tissues was reverse-transcribed using SuperScript II reverse transcriptase (Life Technologies, Inc.) and amplified with PCR to detect cDNA fragments of GLAST, GLT-1, EAAC1, and EAAT4. Primer pairs used were as follows: GLAST, 5′-ATT GTA CAA GTG ACA GCT GCA GAC GCC-3′ and 5′-TTT CCC TGC AAT CAG GAA GAG GAT GCC C-3′ (nt 481–507 and nt 938–912 of canine GLAST, respectively); GLT-1, 5′-GCC ATG GTG TAT TAC ATG TCC ACA ACC A-3′ and 5′-CCA TCC TTG AAC TCC AAG CCC TTC TTG-3′ (corresponding to nt 358–385 and nt 713–687 of rat GLT-1 (2.Pines 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 (1136) Google Scholar)); EAAC1, 5′-TGG GAA ATA TTC CGC AAG CTA GGC CTT-3′ and 5′-TTT CTT CTG CAC AGC GGA AAG TGA CAG G-3′ (corresponding to nt 826–851 and nt 1,039–1,012 of rabbit EAAC1 (3.Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1197) Google Scholar)); and EAAT4, 5′-CAC TCA TTG TCT CCA GCC TGG TCA CAG-3′ and 5′-TCT TGA ACT GTT TGA AGC AGG CCT CCA-3′ (corresponding to nt 317–343 and nt 598–572 of human EAAT4 (4.Fairman W.A. Vandenberg R.J. Arriza J.L. Kavanaugh M.P. Amara S.G. Nature. 1995; 375: 599-603Crossref PubMed Scopus (1013) Google Scholar)). These sequences specific to canine glutamate transporter subtypes were derived from those of cDNA fragments amplified by PCR from canine brain cDNAs using degenerate primers designed according to the sequences of rat GLAST (5′-ACC AC(C/T) ATC ATT GCT GTG GTG-3′ and 5′-GC(A/G) GTC CCA TCC ATG TTA ATG-3′, corresponding to nt 388–408 and nt 1,208–1,188 of rat GLAST (1.Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1097) Google Scholar)), rat GLT-1 (5′- CTG GAT GCT AAG GCT AGT GGC CGC-3′ and 5′-GC(A/G) GTC CCA TCC ATG TTA ATG-3′, corresponding to nt 322–345 and nt 1,202–1,182, respectively), rabbit EAAC1 (5′-GG(A/C/G/T) GAA ATC CTG ATG AGG ATG CTG-3′ and 5′-GC(A/G) GTC CCA TCC ATG TTA ATG-3′, corresponding to nt 166–189 and nt 1,112–1,092, respectively) and human EAAT4 (5′-GG(A/C/G/T) GAA ATC CTG ATG AGG ATG CTG-3′ and 5′-GGG GAA GGG GTT CCG GTG AGT GAC-3′, corresponding to nt 277–300 and nt 1,116–1,093, respectively). Canine glyceraldehyde 3-phosphate dehydrogenase gene transcripts were also amplified as an internal control using primers (5′-TGC TCC TTC TGC TGA TGC CCC CAT-3′ and 5′-TCT GGG TGG CAG TGA TGG CAT GGA-3′) prepared according to the sequence reported by Grone et al. (24.Grone A. Weckmann M.T. Capen C.C. Rosol T.J. Am. J. Vet. Res. 1996; 57: 254-257PubMed Google Scholar). PCR products were analyzed on 2% agarose gel (see Fig. 2) and sequenced to confirm the specificity of the products. The similarities of nucleotide sequences of each PCR product to rat GLAST, rat GLT-1, rabbit EAAC1, and human EAAT4 were 86.7, 86.6, 88.8, and 93.3%, respectively. Adapter-ligated cDNAs were prepared from poly(A)+ RNA from the forebrain cortex of a dog, using a Marathon cDNA Amplification kit (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions. RACE reactions were performed using Advantage Klen Taq DNA polymerase (CLONTECH) with the adapter primers supplied by the manufacturer and gene-specific primers synthesized according to the sequences of the PCR-amplified cDNA fragment of canine brain GLAST. The GLAST-specific primers for 5′- and 3′-RACE were 5′-TGC CCC CCA ATT ACT CCC ATG TCT TCC-3′ (nt 938–912) and 5′-ATT GTA CAA GTG ACA GCT GCA GAC GCC-3′ (nt 481–507), respectively. Nested primers were 5′-TTT CCC TGC AAT CAG GAA GAG GAT GCC C-3′ (nt 900–873) for 5′-RACE and 5′-AAA GTG CCC ATC CAG TCC AAT GAG ACG-3′ (nt 598–624) for 3′-RACE. PCR-amplified fragments were cloned into pCR2.1 by the TA cloning method (Invitrogen, San Diego, CA) and combined into pSPORT1 vector (Life Technologies, Inc., Life Technologies, Inc., Rockville, MD) to create 5′- and 3′-stretched canine brain GLAST cDNA, pcGLAST. A bone marrow GLAST cDNA clone (pcGLASTbm) was also prepared in the same manner. Oocytes (stage V and VI) were isolated from Xenopus laevis under ice-cold anesthesia and defolliculated by treatment with 0.2% collagenase in ND96 (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES/Tris, pH 7.5). The oocytes were microinjected with 1–25 ng of capped synthetic GLAST RNAs (50 nl). GLAST RNAs were transcribed from pcGLAST or pcGLASTbm linearized with BglII immediately downstream from the termination codon or withMluI within the vector sequence using MaxiScript or MegaScript kit with T7 RNA polymerase and a cap analogue (Ambion, Austin, TX). The oocytes were incubated at 19 °C in ND96 containing 1.8 mm CaCl2 100 units/ml penicillin, and 100 μg/ml streptomycin for 36–72 h. In some experiments, cycloheximide (Wako Pure Chemical Industries, Osaka, Japan) was added to the medium at a concentration of 10 μg/ml, and incubation was continued for 12 h. Multiple antigen peptides were synthesized using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase method for the amino acid sequence of the carboxyl-terminal region of canine GLAST (NH2-Asn-Ser-Val-Ile-Glu-Glu-Asn-Glu-Met-Lys-Lys-Pro-Tyr-Gln-Leu-COOH; residues 511–525; see Fig. 1) using a peptide synthesizer (Shimadzu, Kyoto, Japan) at Dr. Y. Takakuwa's laboratory (Tokyo Women's Medical University). New Zealand White rabbits were immunized with 100–200 μg of peptides with Freund's complete adjuvant followed by three successive immunizations with Freund's incomplete adjuvant at 2-week intervals. Antisera were obtained 3 days after intravenous injection of the peptides. Anti-GLAST antibody was purified on an affinity chromatography medium that was prepared by immobilizing synthetic peptides to an N-hydroxysuccinimide-activated HiTrap column (Amersham Pharmacia Biotech) at a concentration of 1 mg/ml packed gel. Antibodies bound to the column were eluted with 0.5 m NaCl, 0.1 m glycine/Tris, pH 2.7, and neutralized immediately with Tris. Red cell ghosts were prepared as described (16.Inaba M. Maede Y. J. Biol. Chem. 1986; 261: 16099-16105Abstract Full Text PDF PubMed Google Scholar). Crude synaptic membranes from brain were prepared according to Kanner (25.Kanner B.I. Biochemistry. 1978; 17: 1207-1211Crossref PubMed Scopus (210) Google Scholar). Membrane proteins were separated by SDS-PAGE (Laemmli's system) on 8% gels (23.Inaba M. Yawata A. Koshino I. Sato K. Takeuchi M. Takakuwa Y. Manno S. Yawata Y. Kanzaki A. Sakai J. Ban A. Ono K. Maede Y. J. Clin. Invest. 1996; 97: 1804-1817Crossref PubMed Scopus (163) Google Scholar). The GLAST polypeptides were detected by immunoblotting using affinity-purified anti-GLAST antibodies and an ECL chemiluminescence detection system (Amersham Pharmacia Biotech). In some experiments, membrane proteins were solubilized in 2% (w/v) CHAPS (Dojin Laboratories, Kumamoto, Japan) and kept on ice for 30 min to induce oligomerization of the GLAST proteins, which leads to efficient detection of the polypeptides with the antibodies. Solubilized proteins were concentrated with ultrafiltration units (Ultrafree MC, 30,000 nominal molecular weight limit; Millipore Corp., Bedford, MA) and subjected to SDS-PAGE followed by immunoblotting. pcGLAST was transcribed and translated using a TnT T7 coupled reticulocyte lysate system (Promega Corp., Madison, WI) with or without canine pancreatic microsomes (Promega) in the presence of [35S]methionine (EXPRE35S35S; 1,175 Ci/mmol; NEN Life Science Products). Translated products were analyzed by SDS-PAGE followed by exposure to Kodak BioMax MR films. Red cell membranes and crude synaptic membranes were deglycosylated using peptide:N-glycosidase F (NEN Life Science Products). Briefly, the membrane proteins (100–150 μg) were solubilized in 0.5% SDS, 1% β-mercaptoethanol at room temperature for 30 min. After the addition of 110 volume of 10% Nonidet P-40 and 0.5m sodium phosphate, pH 7.5, samples were incubated with 2,000 units of peptide:N-glycosidase F at 37 °C for 1 h. Reactions were stopped by the addition of Laemmli's sample buffer and subjected to SDS-PAGE and immunoblotting. Uptake of l-glutamate in canine red cells was measured as described previously (12.Inaba M. Maede Y. J. Biol. Chem. 1984; 259: 312-317Abstract Full Text PDF PubMed Google Scholar, 13.Sato K. Inaba M. Maede Y. Biochim. Biophys. Acta. 1994; 1195: 211-217Crossref PubMed Scopus (9) Google Scholar). Oocytes injected with synthetic RNA were incubated at 19 °C in ND96 medium containing l-[3,4-3H]glutamate (49 Ci/mmol; NEN Life Science Products) and 1.8 mmCaCl2 (100 μl/oocyte). After incubation, oocytes were washed three times with an excessive amount of ice-chilled ND96 medium. Each oocyte was transferred into a 1.5-ml tube and lysed in 200 μl of 1% SDS, and radioactivity was determined using ReadyCap (Beckman, Fullerton, CA). To estimate Na+-independent transport, NaCl was substituted for equimolar choline chloride. Under these conditions, the Na+-dependent component, given by subtraction of the values in the absence of Na+ from those in the presence of Na+, increased linearly for the initial 5 min. The sensitivity of the transporter to the inhibitor was estimated by determining the transport activity for 5 μml-glutamate in the presence of appropriate concentrations of various compounds. A restriction enzyme assay was carried out to determine genotypes for G492S mutation. PCR fragments corresponding to nt 1,427–1,657 of canine GLAST cDNA were amplified using genomic DNA from dogs as the templates. The resulting PCR products were digested with NgoMIV and separated on 4% agarose gel. GLAST mRNA was quantitated by RT-PCR combined with a 5′-nuclease assay or SYBR green detection using the GeneAmp 5700 sequence detection system (Perkin-Elmer Applied Biosystems). A 5′-nuclease assay of GLAST mRNA was carried out with PCR primers 5′-AAT GTG TCG GAA GCC ATG GAG-3′ (nt 646–666) and 5′-TTG ACC CCA TTC ACA GAC CCT-3′ (nt 725–705) in the presence of a TaqMan probe of 5′-ACA AGG ATC ACG GAG GAG TTG ATC CCA G-3′ (nt 673–700) and was normalized with the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA. In our preliminary experiments, PCR amplification of canine brain cDNA generated cDNA fragments corresponding to those of four different glutamate transporter subtypes, GLAST, GLT-1, EAAC1, and EAAT4. Nucleotide sequences of these cDNA fragments from dogs showed high similarities to those from other sources as described under “Experimental Procedures.” However, when the same procedure was applied to cDNA from bone marrow cells, only cDNA fragments corresponding to GLAST were obtained, suggesting that the GLAST protein functions in canine erythroid cells. Primers specific to the “GLAST-like” sequences obtained were prepared, and 5′- and 3′-RACE reactions were carried out using canine brain and bone marrow cDNAs as templates. The 5′- and 3′-RACE products were subcloned and combined. The cDNA clones from the brain (pcGLAST) and bone marrow cells (pcGLASTbm) were both about 3.8 kilobase pairs in length with a 1,629-bp open reading frame encoding a protein of 542 amino acid residues with the theoretical molecular mass of 59,757 Da. The size of the GLAST mRNA was confirmed by Northern blotting, although the signal intensity was very weak for bone marrow mRNA even when more than 10 μg of poly(A)+ RNA was applied (data not shown). The deduced amino acid sequence showed high similarity, over 96%, to human, bovine, and rat GLASTs (Fig.1) and significant but much lesser similarity to the other glutamate transporters, GLT-1, EAAC1, EAAT4, and EAAT5 (50–66%). Thus, recent models for membrane topology of human GLAST (26.Wahle S. Stoffel W. J. Cell Biol. 1996; 135: 1867-1877Crossref PubMed Scopus (47) Google Scholar, 27.Seal R.P. Amara S.G. Neuron. 1998; 21: 1487-1498Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) with 10 or 11 membrane-spanning regions and cytoplasmic localization of NH2 and COOH termini can be adopted to the canine homologue. This prediction supposes two potentialN-glycosylation sites, at Asn206 and Asn216, with N-glycosylation consensus sequences (Asn-X, where X represents any residue except Pro-Ser/Thr) within the putative second extracellular loop. RT-PCR analysis indicated amplification of GLAST cDNA with strong signals in the cerebral cortex, cerebellum, and hippocampus (Fig.2). Amplification was also observed in other tissues and cells except that a very faint band and no bands were obtained in reticulocytes and liver, respectively. It should be emphasized that signals for GLAST cDNA were detected clearly in reticulocytes and liver when the PCR cycles were increased (Fig. 2,right panels). Signals were also detected in all other tissues examined, including colon, spleen, pancreas, thyroid gland, adrenal gland, and testis, with intensities similar to those of reticulocytes and liver (data not shown). Under the PCR conditions employed, no noticeable amplification of the transporter cDNA other than that of GLAST cDNA was observed in bone marrow cells and reticulocytes, although EAAC1 showed a very faint band of PCR products in bone marrow after extended PCR cycles. These results demonstrated a ubiquitous expression of GLAST transcripts in a variety of cells and tissues in dogs and indicated that canine erythroid cells contained the GLAST mRNA but not the transcripts of other glutamate transporter genes. Oocytes injected with synthetic RNA of canine GLAST showed high affinity Na+-dependent glutamate uptake that was completely abolished when the extracellular Na+was replaced by choline. The Na+-dependent uptake was dominated by a saturable component obeying Michaelis-Menten kinetics (Fig. 3 A). TheK m value for l-glutamate obtained from a Lineweaver-Burk plot (Fig. 3 B) was 36.3 μm. This value was slightly higher than that estimated for the uptake in canine red cells at 37 °C (7–14 μm; Refs. 12.Inaba M. Maede Y. J. Biol. Chem. 1984; 259: 312-317Abstract Full Text PDF PubMed Google Scholar and 13.Sato K. Inaba M. Maede Y. Biochim. Biophys. Acta. 1994; 1195: 211-217Crossref PubMed Scopus (9) Google Scholar), whereas lower affinity was reported for other mammalian GLAST homologues in oocytes (70–80 μm) (1.Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1097) Google Scholar, 28.Tanaka K. Neurosci. Lett. 1993; 159: 183-186Crossref PubMed Scopus (74) Google Scholar, 29.Kawakami H. Tanaka K. Nakayama T. Inoue K. Nakamura S. Biochem. Biophys. Res. Commun. 1994; 199: 171-176Crossref PubMed Scopus (55) Google Scholar, 30.Inoue K. Sakaitani M. Shimada S. Tohyama M. Mol. Brain Res. 1995; 28: 343-348Crossref PubMed Scopus (24) Google Scholar). Several structural analogues of l-glutamate were tested for their inhibitory effects on glutamate transport to compare the pharmacological properties of canine GLAST and canine red cells (TableI). Potent inhibition of the glutamate uptake was observed in response to threo-3-hydroxyaspartate,l-glutamate, and l- and d-aspartate but not by d-glutamate. These were the properties common to all the brain glutamate transporters (31.Kanai Y. Curr. Opin. Cell Biol. 1997; 9: 565-572Crossref PubMed Scopus (130) Google Scholar). Dihydrokainate andl-cysteine, which are selective inhibitors for human GLT-1 (32.Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar) and human EAAC1 (33.Zerangue N. Kavanaugh M.P. J. Physiol. 1996; 493: 419-423Crossref PubMed Scopus (174) Google Scholar), respectively, poorly inhibited the glutamate transport by canine GLAST. Thus, the responses of canine GLAST expressed in the oocytes were consistent with those of the glutamate transport system in canine red cells (Table I).Table ICross-inhibition with structural analogues of glutamate transport by canine GLAST expressed in oocytesInhibitorsUptake (percentage of control)OocytesRed cellsaReference data for dog red cells at 37 °C determined in our previous study (13) except forl-cysteine.%%None100.0 ± 21.8100.0 ± 5.0l-Glutamate46.3 ± 24.022.3 ± 0.3d-Glutamate87.8 ± 21.376.7 ± 11.8l-Aspartate43.9 ± 16.920.3 ± 0.9d-Aspartate61.5 ± 27.953.2 ± 2.2threo-3-Hydroxyaspartate15.7 ± 5.616.6 ± 0.4l-Cysteinesulfinate43.5 ± 6.020.6 ± 1.0Dihydrokainate (500 μm)73.1 ± 21.256.4 ± 11.7 (1 mm)l-Cysteine (500 μm)73.3 ± 10.788.4 ± 1.0The uptake of 5 μm l-[3H]glutamate was measured in oocytes expressing canine GLAST at 19 °C for 5 min in ND96 medium containing a 25 μm concentration of each analogue. Values represent the mean ± S.D. (n = 6).a Reference data for dog red cells at 37 °C determined in our previous study (13.Sato K. Inaba M. Maede Y. Biochim. Biophys. Acta. 1994; 1195: 211-217Crossref PubMed Scopus (9) Google Scholar) except forl-cysteine. Open table in a new tab The uptake of 5 μm l-[3H]glutamate was measured in oocytes expressing canine GLAST at 19 °C for 5 min in ND96 medium containing a 25 μm concentration of each analogue. Values represent the mean ± S.D. (n = 6). Affinity-purified antibodies to the synthetic peptide of GLAST reacted with 60-kDa proteins in membranes from the cerebral cortex, while the antibodies immunospecifically recognized polypeptides with an apparent molecular mass of 66 kDa in red cell membranes, as documented in Fig. 4 A(Membranes). When the brain membranes were solubilized with CHAPS, the distinct higher molecular mass signals at 120 kDa appeared on the immunoblot (Fig. 4 A, CHAPS extracts) as reported for rat GLAST (34.Haugeto O. Ullensvang K. Levy L.M.