Cloning and Characterization of a Functional Human γ-Aminobutyric Acid (GABA) Transporter, Human GAT-2
2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês
10.1074/jbc.m702111200
ISSN1083-351X
AutoresB. C. Christiansen, Anne‐Kristine Meinild, Anders A. Jensen, Hans Bräuner‐Osborne,
Tópico(s)Biochemical effects in animals
ResumoPlasma membrane γ-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human. The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable and dependent on both Na+ and Cl–. Pharmacologically the transporter is distinct from the other human GABA transporters and similar to rat GAT-2 and mouse GAT3 with high sensitivity toward GABA and β-alanine. Furthermore the GABA transport inhibitor (S)-SNAP-5114 displayed some inhibitory activity at the transporter. Expression analysis by reverse transcription-PCR showed that GAT-2 mRNA is present in human brain, kidney, lung, and testis. The finding of the human GAT-2 demonstrates for the first time that the four plasma membrane GABA transporters identified in several mammalian species are all conserved in human. Furthermore the availability of human GAT-2 enables the use of all human clones of the GABA transporters in drug development programs and functional characterization of novel inhibitors of GABA transport. Plasma membrane γ-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human. The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable and dependent on both Na+ and Cl–. Pharmacologically the transporter is distinct from the other human GABA transporters and similar to rat GAT-2 and mouse GAT3 with high sensitivity toward GABA and β-alanine. Furthermore the GABA transport inhibitor (S)-SNAP-5114 displayed some inhibitory activity at the transporter. Expression analysis by reverse transcription-PCR showed that GAT-2 mRNA is present in human brain, kidney, lung, and testis. The finding of the human GAT-2 demonstrates for the first time that the four plasma membrane GABA transporters identified in several mammalian species are all conserved in human. Furthermore the availability of human GAT-2 enables the use of all human clones of the GABA transporters in drug development programs and functional characterization of novel inhibitors of GABA transport. γ-Aminobutyric acid (GABA) 2The abbreviations used are: GABA, γ-aminobutyric acid; BGT-1, betaine/GABA transporter-1; l-DABA, l-2,4-diamino-n-butyric acid; DAPA, dl-2,3-diaminopropionic acid; EF1502, N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol; FMP, FLIPR membrane potential; GAT, GABA transporter; MTC, multiple tissue cDNA; NCBI, National Center for Biotechnology Information; SLC6A13, solute carrier family 6 (neurotransmitter transporter, GABA), member 13; THPO, 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol; m, mouse; h, human; r, rat; MES, 4-morpholineethanesulfonic acid. 2The abbreviations used are: GABA, γ-aminobutyric acid; BGT-1, betaine/GABA transporter-1; l-DABA, l-2,4-diamino-n-butyric acid; DAPA, dl-2,3-diaminopropionic acid; EF1502, N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol; FMP, FLIPR membrane potential; GAT, GABA transporter; MTC, multiple tissue cDNA; NCBI, National Center for Biotechnology Information; SLC6A13, solute carrier family 6 (neurotransmitter transporter, GABA), member 13; THPO, 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol; m, mouse; h, human; r, rat; MES, 4-morpholineethanesulfonic acid. is the major inhibitory neurotransmitter in the mammalian central nervous system (1Krnjević K. 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Reith M.E. Quick M.W. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 519-531Crossref PubMed Scopus (335) Google Scholar). The transport process of these transporters is electrogenic because Na+ (and Cl–) is translocated across the membrane together with the respective substrates (7Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (321) Google Scholar, 19Gether U. Andersen P.H. Larsson O.M. Schousboe A. Trends Pharmacol. Sci. 2006; 27: 375-383Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). As an example, the co-transport of one GABA, two Na+, and one Cl– has been reported for human (h) GAT-1 (20Loo D.D. Eskandari S. Boorer K.J. Sarkar H.K. Wright E.M. J. Biol. Chem. 2000; 275: 37414-37422Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 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Mouse, rat, and human BGT-1 is expressed in both the brain and periphery (10López-Corcuera B. Liu Q.R. Mandiyan S. Nelson H. Nelson N. J. Biol. Chem. 1992; 267: 17491-17493Abstract Full Text PDF PubMed Google Scholar, 14Borden L.A. Smith K.E. Gustafson E.L. Branchek T.A. Weinshank R.L. J. Neurochem. 1995; 64: 977-984Crossref PubMed Scopus (129) Google Scholar, 16Rasola A. Galietta L.J. Barone V. Romeo G. Bagnasco S. FEBS Lett. 1995; 373: 229-233Crossref PubMed Scopus (56) Google Scholar, 17Burnham C.E. Buerk B. Schmidt C. Bucuvalas J.C. Biochim. Biophys. Acta. 1996; 1284: 4-8Crossref PubMed Scopus (38) Google Scholar) and believed to be involved in osmoregulation (18Chen N.H. Reith M.E. Quick M.W. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 519-531Crossref PubMed Scopus (335) Google Scholar) but has recently also been suggested to play a role in the control of epilepsy (41Clausen R.P. Frølund B. Larsson O.M. Schousboe A. Krogsgaard-Larsen P. White H.S. Neurochem. Int. 2006; 48: 637-642Crossref PubMed Scopus (36) Google Scholar, 42Schousboe A. Larsson O.M. Sarup A. White H.S. Eur. J. Pharmacol. 2004; 500: 281-287Crossref PubMed Scopus (32) Google Scholar, 43White H.S. Watson W.P. Hansen S.L. Slough S. Perregaard J. Sarup A. Bolvig T. Petersen G. Larsson O.M. Clausen R.P. Frølund B. Falch E. Krogsgaard-Larsen P. Schousboe A. J. Pharmacol. Exp. Ther. 2005; 312: 866-874Crossref PubMed Scopus (70) Google Scholar). Rat (r) GAT-2 and mouse (m) GAT3 is similarly found in the brain as well as in the periphery where it is abundantly expressed in kidney and liver (11Borden L.A. Smith K.E. Hartig P.R. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1992; 267: 21098-21104Abstract Full Text PDF PubMed Google Scholar, 13Liu Q.R. López-Corcuera B. Mandiyan S. Nelson H. Nelson N. J. Biol. Chem. 1993; 268: 2106-2112Abstract Full Text PDF PubMed Google Scholar, 36Ikegaki N. Saito N. Hashima M. Tanaka C. Brain Res. Mol. Brain Res. 1994; 26: 47-54Crossref PubMed Scopus (115) Google Scholar, 39Durkin M.M. Smith K.E. Borden L.A. Weinshank R.L. Branchek T.A. Gustafson E.L. Brain Res. Mol. Brain Res. 1995; 33: 7-21Crossref PubMed Scopus (173) Google Scholar, 44Jursky F. Nelson N. J. Neurosci. Res. 1999; 55: 394-399Crossref PubMed Scopus (23) Google Scholar). In contrast to the four identified plasma membrane GABA transporter subtypes in mouse and rat, only three have been characterized in human. These include hGAT-1, hBGT-1, and hGAT-3, whereas the human ortholog of mGAT3 and rGAT-2 has remained enigmatic and typically is referred to as "not cloned" in the literature (35Dalby N.O. Eur. J. Pharmacol. 2003; 479: 127-137Crossref PubMed Scopus (150) Google Scholar, 45Sarup A. Larsson O.M. Schousboe A. Curr. Drug Targets CNS Neurol. Disord. 2003; 2: 269-277Crossref PubMed Scopus (102) Google Scholar). However, in 2001, the cDNA supposedly encoding for the hGAT-2 was reported to have been cloned, although functional uptake of [3H]GABA in mammalian cells transiently transfected with this cDNA could not be demonstrated (46Gong Y. Zhang M. Cui L. Minuk G.Y. Can. J. Physiol. Pharmacol. 2001; 79: 977-984Crossref PubMed Scopus (16) Google Scholar). Bioinformatics analysis of this putative hGAT-2 sequence revealed that it was likely to be an incomplete cDNA sequence with several truncations. In the present study, we cloned the full-length hGAT-2 and characterized the pharmacology of the transporter in several functional assays. Materials—GlutaMAX-I Dulbecco's modified Eagle's medium, dialyzed fetal bovine serum, penicillin, streptomycin, Hanks' balanced salt solution, and bovine serum albumin were purchased from Invitrogen. All buffer reagents were obtained from Sigma-Aldrich. [2,3-3H]GABA (specific radioactivity, 27.6 Ci/mmol) and d-[2,3-3H]Asp (specific radioactivity, 40.0 Ci/mmol) were purchased from GE Healthcare. β-Alanine, taurine, l-2,4-diamino-n-butyric acid (l-DABA), quinidine, NNC-711, and (S)-SNAP-5114 were purchased from Sigma-Aldrich. GABA was obtained from Fluka Chemie AG, Buchs SG, (Dübendorf, Switzerland), betaine was from B.A.S. Synteselaboratorium, nipecotic acid was from Aldrich, and dl-2,3-diaminopropionic acid (DAPA) was from TCI Europe nv (Zwijndrecht, Belgium). The following compounds were synthesized in house: 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol (THPO) (47Sauerberg P. Larsen J.J. Falch E. Krogsgaard-Larsen P. J. Med. Chem. 1986; 29: 1004-1009Crossref PubMed Scopus (27) Google Scholar), guvacine (48McElvain S.M. Stork G. J. Am. Chem. Soc. 1946; 68: 1049-1053Crossref PubMed Scopus (21) Google Scholar), and N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) (49Clausen R.P. Moltzen E.K. Perregaard J. Lenz S.M. Sanchez C. Falch E. Frølund B. Bolvig T. Sarup A. Larsson O.M. Schousboe A. Krogsgaard-Larsen P. Bioorg. Med. Chem. 2005; 13: 895-908Crossref PubMed Scopus (82) Google Scholar). Cloning of hGAT-2—The I.M.A.G.E. cDNA clone 4612245 was purchased, and subsequent sequencing revealed the presence of the full-length nucleotide sequence encoding for the open reading frame of hGAT-2. The cDNA of hGAT-2 was amplified by PCR using the forward primer 5′-gggatggatagcagggtctc-3′ and the reverse primer 5′-ctagcagtgagactctagctc-3′ and subcloned into the mammalian pcDNA5 vector according to the protocol of the manufacturer (pcDNA5/FRT/V5-His TOPO® TA Expression kit, Invitrogen). The sequence of the cDNA and the absence of mutations were confirmed by automated DNA sequencing. Bioinformatics Analysis of the Protein Sequence of hGAT-2—An alignment of hGAT-2 with the previously reported sequences of hGAT-2 (46Gong Y. Zhang M. Cui L. Minuk G.Y. Can. J. Physiol. Pharmacol. 2001; 79: 977-984Crossref PubMed Scopus (16) Google Scholar), mGAT3 (13Liu Q.R. López-Corcuera B. Mandiyan S. Nelson H. Nelson N. J. Biol. Chem. 1993; 268: 2106-2112Abstract Full Text PDF PubMed Google Scholar), and rGAT-2 (11Borden L.A. Smith K.E. Hartig P.R. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1992; 267: 21098-21104Abstract Full Text PDF PubMed Google Scholar) was performed using the ClustalW alignment program available at the home page maintained by The European Bioinformatics Institute. Transmembrane segments in the hGAT-2 protein were identified by the hidden Markov model for prediction of transmembrane helices (50Sonnhammer E.L. von Heijne G. Krogh A. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar). The algorithm is publicly accessible at the Center for Biological Sequence Analysis, Technical University of Denmark through internet services. Furthermore a hydrophobicity analysis using the TMpred program was performed. This algorithm is based on the statistical analysis of TMbase, a data base of naturally occurring transmembrane proteins (51Hofmann K. Stoffel W. Biol. Chem. Hoppe-Seyler. 1993; 47: 166Google Scholar) and is available at the home page maintained by Swiss EMBnet. The overall sequence identity of the predicted amino acid sequence of hGAT-2 to other related GABA transporters was examined by searching the protein data base at the National Center for Biotechnology Information (NCBI) using the BLASTp algorithm. Expression Analysis—The expression pattern of hGAT-2 mRNA was examined by reverse transcription-PCR using human multiple tissue cDNA (MTC™) panels according to the protocol of the manufacturer (Clontech). The cDNA was amplified by PCR using the forward primer 5′-atggatagcagggtctcaggcacaaccagtaatgg-3′ and the reverse primer 5′-attctcagaggtaccattcagggagccgttgg-3′. The primers are complementary to exons 1 and 4, respectively, and PCR with the two primers resulted in a specific band of 533 nucleotides. PCR was performed using Taq polymerase as described by the manufacturer (Promega, Madison, WI) and a PTC-100 thermal cycler (MJ Research, Waltham, MA). The reactions were heated to 95 °C for 2 min and then cycled 35 times at 95 °C for 1 min, 65 °C for 30 s, and 72 °C for 1 min. All reactions were carried out in parallel and were run on a 1% agarose gel containing SYBR Safe™ (Invitrogen). Amplification of a 983-base pair fragment from human glyceraldehyde-3-phosphate dehydrogenase was used as a control. Expression of hGAT-2 in Xenopus laevis Oocytes—The hGAT-2 and the mGAT3 cDNAs were subcloned by PCR into an expression vector (pEXP-SML) containing 5′- and 3′-untranslated regions of the Xenopus β-globin gene and a poly(A) signal for optimal expression in oocytes using the GATEWAY® technology (Invitrogen). The cDNA was linearized downstream of the poly(A) signal and in vitro transcribed with the T7 RNA polymerase using the T7-Message Machine® kit from Ambion (Ambion, Inc., Austin, TX). 50 ng of cRNA was injected into defolliculated stage 5-6 X. laevis oocytes, prepared as described in Meinild et al. (52Meinild A. Klaerke D.A. Loo D.D. Wright E.M. Zeuthen T. J. Physiol. 1998; 508: 15-21Crossref PubMed Scopus (159) Google Scholar). The oocytes were incubated in Kulori medium (90 mm NaCl, 1 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.4) at 19 °C for 3–5 days before experiments were performed. Electrophysiology—The two-electrode voltage clamp method was used to control the membrane potential and monitor the whole cell current in oocytes expressing hGAT-2. The recordings were performed at room temperature with a Dagan Clampator (Dagan Corp., Minneapolis, MN) interfaced to a personal computer using a DigiData 1320 analog/digital converter and pCLAMP 9 (Axon Instruments at Molecular Devices, Sunnyvale, CA). For continuous current measurements, the membrane potential was held at –50 mV, and the currents were low pass-filtered at 1 Hz and sampled at 10 Hz. To obtain steady-state current/voltage relationships the membrane potential was held at –50 mV and jumped to test potentials ranging from +50 to –150 mV in 20-mV increments for 200 ms. Currents were low pass-filtered at 500 Hz and sampled at 2 kHz. In general, the experimental chamber was continuously perfused by a NaCl solution (100 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, pH 7.4). In Na+ substitution experiments, Na+ was equimolarly replaced with choline ions, and in experiments with low concentrations of Cl– (6 mm), NaMES was used instead of NaCl. In experiments with low Cl– concentrations, the reference electrode was connected to the experimental chamber via an agar bridge (3% agar in 3 m KCl). Cell Culture and Transfections—tsA201 cells (a transformed human embryonic kidney 293 cell line) (53Chahine M. Bennett P.B. George Jr., A.L. Horn R. Pfluegers Arch. Eur. J. Physiol. 1994; 427: 136-142Crossref PubMed Scopus (108) Google Scholar) were cultured in GlutaMAX-I Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The constructs encoding the hGAT-2 and the human excitatory amino acid transporter hEAAT3 were transiently transfected into cells using PolyFect according to the protocol of the manufacturer (Qiagen, West Sussex, UK), and the functional assays were performed 36–48 h later. [3H]GABA and d-[3H]Asp Uptake Assays—tsA201 cells transfected with hGAT-2-pcDNA5 or hEAAT3-pcDNA3 were split into poly-d-lysine-coated white 96-well plates (BD Biosciences). The next day, the medium was removed, and cells were washed with 100 μl of assay buffer (Hanks' balanced salt solution supplemented with 20 mm HEPES, 1 mm CaCl2, and 1 mm MgCl2, pH 7.4). Then 75 μl of assay buffer supplemented with [3H]GABA or d-[3H]Asp and various concentrations of the test compounds was added to each well, and the plate was incubated at 37 °C for 3 min. Then the cells were washed with 3 × 100 μl of ice-cold assay buffer, and 150 μl of Microscint™20 scintillation fluid (PerkinElmer Life Sciences) was added to each well. The plate was shaken for at least 1 h and counted in a Packard TopCount microplate scintillation counter. In the saturation experiments, a [3H]GABA concentration up to 100 nm was used, and to measure transport at higher concentrations the radioligands were diluted with the corresponding "cold" ligand (GABA). Nonspecific transport was determined in the presence of 3 mm GABA. In the competition transport experiments, either 30 nm [3H]GABA (in the experiments with hGAT-2) or 30 nmd-[3H]Asp (in the experiments with hEAAT3) was used as tracer concentration. The [3H]GABA competition curves were constructed based on measurements obtained typically for eight different concentrations of the test compounds. The following maximal concentrations of the test compounds were applied: GABA, 3 mm; DAPA, 1 mm; β-alanine, 3 mm; (S)-SNAP-5114, 500 μm; EF1502, 250 μm; nipecotic acid, 10 mm; l-DABA, 3 mm; quinidine, 1 mm; guvacine, 10 mm; NNC-711, 1.6 mm; THPO, 10 mm; taurine, 10 mm; and betaine, 10 mm. The FLIPR® Membrane Potential (FMP) Assay—The test compounds were characterized functionally in the FMP assay (Molecular Devices, Crawley, UK) essentially as described previously (54Jensen A.A. Bräuner-Osborne H. Biochem. Pharmacol. 2004; 67: 2115-2127Crossref PubMed Scopus (64) Google Scholar). Briefly tsA201 cells transfected with hGAT-2-pcDNA5 were split into poly-d-lysine-coated black clear bottom 96-well plates (BD Biosciences). The next day, the culture medium was removed, and the cells were washed with 100 μl of assay buffer (same buffer as used in the [3H]GABA uptake assay). In the substrate experiments, 100 μl of assay buffer supplemented with FMP assay dye was added to each well, and the plate was incubated at 37 °C for 30 min. The plate was assayed at 37 °C in a NOVOstar™ plate reader (BMG Labtechnologies, Offenburg, Germany) measuring emission at 560 nm caused by excitation at 530 nm before and up to 1 min after addition of 25 μl of substrate solution (the substrate was dissolved in assay buffer). Inhibition experiments were performed similarly except that a mixture of 50 μl of FMP assay dye solution (2× final concentration in assay buffer) and 50 μlof inhibitor solution (2× final concentration in assay buffer) was incubated at 37 °C for 30 min, and the plate was assayed by addition of 25 μl of GABA solution (assay concentration of GABA, 50 μm). The experiments were performed in triplicate at least three times for each test compound. The concentration-response curves for the substrates and the concentration-inhibition curves for the inhibitors were constructed based on the maximal responses obtained for the various concentrations of the respective compounds. For generation of concentration-response curves the following maximal concentrations of the test compounds were applied: GABA, 3 mm; β-alanine, 3 mm; l-DABA, 1 mm; nipecotic acid, 10 mm; guvacine, 3 mm; taurine, 10 mm; betaine, 3 mm; and THPO, 3 mm. Data Analysis—All data were analyzed using Prism 4.0b (GraphPad Software, San Diego, CA). For experiments with X. laevis oocytes, the transporter-specific substrate-induced current (Isubstrate) was obtained from the difference between the currents in NaCl ± substrate. For steady-state kinetic analysis, the Isubstrate was measured at various membrane potentials and external substrate concentrations, and at each voltage the Isubstrate versus concentration of substrate relations were fitted to the Michaelis-Menten equation I = (Imax × [S])/([S] + K0.5) where [S] is the substrate concentration, Imax is the maximal current for saturating [S], and the half-maximal concentration, K0.5, is the substrate concentration giving rise to 50% of Imax. The K0.5 for GABA and β-alanine was obtained at 100 mm external Na+, varying the GABA or β-alani
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