AtGAT1, a High Affinity Transporter for γ-Aminobutyric Acid in Arabidopsis thaliana

Functional characterization of Arabidopsis thaliana GAT1 in heterologous expression systems, i.e. Saccharomyces cerevisiae and Xenopus laevis oocytes, revealed that AtGAT1 (At1g08230) codes for an H+-driven, high affinity γ-aminobutyric acid (GABA) transporter. In addition to GABA, other ω-aminofatty acids and butylamine are recognized. In contrast to the most closely related proteins of the proline transporter family, proline and glycine betaine are not transported by AtGAT1. AtGAT1 does not share sequence similarity with any of the non-plant GABA transporters described so far, and analyses of substrate selectivity and kinetic properties showed that AtGAT1-mediated transport is similar but distinct from that of mammalian, bacterial, and S. cerevisiae GABA transporters. Consistent with a role in GABA uptake into cells, transient expression of AtGAT1/green fluorescent protein fusion proteins in tobacco protoplasts revealed localization at the plasma membrane. In planta, AtGAT1 expression was highest in flowers and under conditions of elevated GABA concentrations such as wounding or senescence. Functional characterization of Arabidopsis thaliana GAT1 in heterologous expression systems, i.e. Saccharomyces cerevisiae and Xenopus laevis oocytes, revealed that AtGAT1 (At1g08230) codes for an H+-driven, high affinity γ-aminobutyric acid (GABA) transporter. In addition to GABA, other ω-aminofatty acids and butylamine are recognized. In contrast to the most closely related proteins of the proline transporter family, proline and glycine betaine are not transported by AtGAT1. AtGAT1 does not share sequence similarity with any of the non-plant GABA transporters described so far, and analyses of substrate selectivity and kinetic properties showed that AtGAT1-mediated transport is similar but distinct from that of mammalian, bacterial, and S. cerevisiae GABA transporters. Consistent with a role in GABA uptake into cells, transient expression of AtGAT1/green fluorescent protein fusion proteins in tobacco protoplasts revealed localization at the plasma membrane. In planta, AtGAT1 expression was highest in flowers and under conditions of elevated GABA concentrations such as wounding or senescence. γ-Aminobutyric acid (GABA) 3The abbreviations used are: GABA, γ-aminobutyric acid; AAP, amino acid permease; ATF, amino acid transporter family; GAT, GABA transporter; GabP, GABA permease; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; HOMOPIPES, homopiperazine-1,4-bis(2-ethanesulfonic acid); ProT, proline transporter.3The abbreviations used are: GABA, γ-aminobutyric acid; AAP, amino acid permease; ATF, amino acid transporter family; GAT, GABA transporter; GabP, GABA permease; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; HOMOPIPES, homopiperazine-1,4-bis(2-ethanesulfonic acid); ProT, proline transporter. is a four-carbon non-protein amino acid present in prokaryotes and eukaryotes. Although GABA was discovered in 1949 as a constituent of potato tubers, research on GABA metabolism and transport advanced much faster in the animal system as GABA turned out to be the most abundant inhibitory neurotransmitter in the central nervous system (1Steward F.C. Thompson J.F. Dent C.E. Science. 1949; 110: 439-440Google Scholar). Uptake of GABA into neurons and glia has been investigated in detail and shown to be mediated by Na+-dependent and Cl--facilitated GABA transporters (GATs), thus regulating concentration and duration of the neurotransmitter GABA in the synapse (2Borden L.A. Neurochem. Int. 1996; 29: 335-356Crossref PubMed Scopus (518) Google Scholar, 3Chen N.-H. Reith M.A. Quick M. Pflugers Arch. Eur. J. Physiol. 2004; 447: 519-531Crossref PubMed Scopus (337) Google Scholar). In addition to its function as a neurotransmitter, GABA plays a role in the development of the nervous system, influencing proliferation, migration, and differentiation (4Owens D.F. 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Res. 1997; 101: 403-409Crossref Scopus (69) Google Scholar, 9Solomon P.S. Oliver R.P. Planta. 2002; 214: 414-420Crossref PubMed Scopus (95) Google Scholar), although other functions such as GABA synthesis for pH regulation in Escherichia coli and for normal oxidative stress tolerance in S. cerevisiae have also been postulated (10Coleman S.T. Fang T.K. Rovinsky S.A. Turano F.J. Moye-Rowley W.S. J. Biol. Chem. 2001; 276: 244-250Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 11Yohannes E. Barnhart D.M. Slonczewski J.L. J. Bacteriol. 2004; 186: 192-199Crossref PubMed Scopus (83) Google Scholar). Much less is known about the role of GABA and its transport across the plasma membrane in plants. GABA rapidly accumulates under various stress conditions such as low temperature, mechanical stimulation, and oxygen deficiency (12Shelp B.J. Bown A.W. McLean M.D. Trends Plant Sci. 1999; 4: 446-452Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 13Bouché N. Fromm H. Trends Plant Sci. 2004; 9: 110-115Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar). As in other organisms, GABA is synthesized in plants primarily by decarboxylation of glutamate and degraded via succinic semialdehyde to succinate, a pathway that is also called the GABA shunt (12Shelp B.J. Bown A.W. McLean M.D. Trends Plant Sci. 1999; 4: 446-452Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar). Alternatively, succinic semialdehyde can be further catabolized to γ-hydroxybutyrate (14Breitkreuz K.E. Allan W.L. Van Cauwenberghe O.R. Jakobs C. Talibi D. André B. Shelp B.J. J. Biol. Chem. 2003; 278: 41552-41556Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In plants GABA and the GABA shunt have been discussed as important for regulation of cytosolic pH, nitrogen storage and metabolism, protection against oxidative stress, development, and deterrence of insects (12Shelp B.J. Bown A.W. McLean M.D. Trends Plant Sci. 1999; 4: 446-452Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 13Bouché N. Fromm H. Trends Plant Sci. 2004; 9: 110-115Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 15Bouché N. Fait A. Bouchez D. Moller S.G. Fromm H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6843-6848Crossref PubMed Scopus (330) Google Scholar). GABA might also act as a compatible solute, and more recently its involvement in pollen tube guidance has been demonstrated, suggesting a role in intercellular signaling in plants (13Bouché N. Fromm H. Trends Plant Sci. 2004; 9: 110-115Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 16Bouché N. Lacombe B. Fromm H. Trends Cell Biol. 2003; 13: 607-610Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 17Palanivelu R. Brass L. Edlund A.F. Preuss D. Cell. 2003; 114: 47-59Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). Such functions require both intra- and intercellular transport of GABA. Indeed, both cellular and vascular transport of GABA have been documented in physiological experiments (18Servaites J.C. Schrader L.E. Jung D.M. Plant Physiol. 1979; 64: 546-550Crossref PubMed Google Scholar, 19Chung I. Bown A.W. Shelp B.J. Plant Physiol. 1992; 99: 659-664Crossref PubMed Scopus (50) Google Scholar, 20van Dongen J.T. Schurr U. Pfister M. Geigenberger P. Plant Physiol. 2003; 131: 1529-1543Crossref PubMed Scopus (173) Google Scholar). So far, only transporters mediating low affinity uptake of GABA (Km in the millimolar range) have been identified in plants. These GABA transporters (AtAAP3, ProTs), which belong to the amino acid/auxin transporter (AAAP) or amino acid transporter (ATF) superfamily (21Young G.B. Jack D.L. Smith D.W. Saier Jr., M.H. Biochim. Biophys. Acta. 1999; 1415: 306-322Crossref PubMed Scopus (121) Google Scholar, 22Wipf D. Ludewig U. Tegeder M. Rentsch D. Koch W. Frommer W.B. Trends Biochem. Sci. 2002; 27: 139-147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), might not transport GABA in planta, as their affinity for amino acids (AtAAP3) (23Breitkreuz K.E. Shelp B.J. Fischer W.N. Schwacke R. Rentsch D. FEBS Lett. 1999; 450: 280-284Crossref PubMed Scopus (93) Google Scholar, 24Fischer W.N. Loo D.D. Koch W. Ludewig U. Boorer K.J. Tegeder M. Rentsch D. Wright E.M. Frommer W.B. Plant J. 2002; 29: 717-731Crossref PubMed Scopus (167) Google Scholar) or for the compatible solutes proline and glycine betaine (ProTs) (23Breitkreuz K.E. Shelp B.J. Fischer W.N. Schwacke R. Rentsch D. FEBS Lett. 1999; 450: 280-284Crossref PubMed Scopus (93) Google Scholar, 25Grallath S. Weimar T. Meyer A. Gumy C. Suter-Grotemeyer M. Neuhaus J.M. Rentsch D. Plant Physiol. 2005; 137: 117-126Crossref PubMed Scopus (134) Google Scholar), respectively, are considerably higher than for GABA. In this study we have reported the identification and characterization of the first high affinity GABA transporter from Arabidopsis, designated AtGAT1. Characterization in heterologous expression systems showed that kinetic properties and substrate selectivity of AtGAT1 are similar but distinct from mammalian, bacterial, and S. cerevisiae GABA transporters described so far. Plant Material, Growth Conditions, and Stress Treatment—Arabidopsis thaliana L. ecotype Col-0 was grown in soil in a growth chamber at 22 °C/18 °C, 65% humidity, and 16 h of light. For induction of wounding response, rosette leaves of 4–5-week-old plants were wounded by scratching them with tweezers. Two 1–1.5-cm-long scratches parallel to the midrib were made, and wounded leaves were harvested 2, 4, and 24 h after wounding. Low and high temperature treatments were performed by keeping plants in the dark at 4 and 37 °C, respectively; anoxic stress was applied by submerging whole Arabidopsis plants in water. Leaves were harvested 2, 4, and 24 h after onset of the treatment. For dark induction of senescence, green leaves were excised and incubated on moistened filter paper for 3 or 6 days in the dark. Alternatively, senescence of attached leaves was assayed by harvesting two batches of yellowing leaves of different stages of senescence from the same plants. ∼20% of the leaf area was yellowing in stage I senescent leaves, whereas ∼50% of the leaf area was yellowing in stage II leaves. Chlorophyll Extraction—Plant material was extracted three times with 80% acetone containing 1 μm KOH, and the chlorophyll content of the extract was measured spectrophotometrically (26Strain H.H. Cope B.T. Svec W.A. Methods Enzymol. 1971; 23: 452-476Crossref Scopus (235) Google Scholar). Determination of GABA Concentration—150–200 mg of plant material was extracted with 500 μl of a mixture of methanol:chloroform: water (12:5:3). After centrifugation, the supernatant was recovered and 188 μl of water and 125 μl of chloroform were added. The upper phase of the mixture was dried at room temperature using a speed vacuum apparatus. The pellet was dissolved in 200 μl of water and 300 μl of acetonitrile, and phases were separated by centrifugation. The supernatant was dried, dissolved in 200 μl of water, and purified using a Sep-Pak Vac 1cc C18 cartridge (Waters, Milford, MA). The eluate was dried, and GABA content was measured by high performance liquid chromatography using a modified protocol according to Bidlingmeyer et al. (27Bidlingmeyer B.A. Cohen S.A. Tarvin T.L. J. Chromatogr. 1984; 336: 93-104Crossref PubMed Scopus (2118) Google Scholar). DNA and RNA Work—The AtGAT1-cDNA was isolated by RT-PCR using primers 5′-ACTTATAAAAGTGAGTAGCACC-3′, 5′-CTCACTTTGCTTTGCATGTTC-3′ and RNA extracted from flowers of A. thaliana L. ecotype Col-0 as template. The AtGAT1-cDNA was cloned in the EcoRV site of pSK and verified by sequencing. For S. cerevisiae complementation assays the AtGAT1-cDNA was transferred into pDR196 using PstI and XhoI (28Rentsch D. Laloi M. Rouhara I. Schmelzer E. Delrot S. Frommer W.B. FEBS Lett. 1995; 370: 264-268Crossref PubMed Scopus (277) Google Scholar). The cDNA of At5g41800 was isolated by RT-PCR using primers 5′-TATATAAATCATTCACTTGTAG-3′, 5′-TTTATGTTTTTATCACACTATC-3′ and RNA extracted from flowers of A. thaliana L. ecotype Col-0 as template. The At5g41800-cDNA was cloned in the SmaI site of pDR196 and verified by sequencing. For translational fusions with GFP, the open reading frame of the AtGAT1 cDNA was amplified by PCR and cloned in pUC18-spGFP6 and pUC18-GFP5Tsp. 4M. Suter Grotemeyer and D. Rentsch, unpublished information. AtGAT1-GFP fusion: 5′-CTAGCTAGCATGGGTGGAGAAGAGAGATC-3′, 5′-GAAGATCTACATCCGCAAACAACTTATAC-3′ (open reading frame cloned into SpeI/BglII site). GFP-AtGAT1 fusion: 5′-CTAGCTAGCATGGGTGGAGAAGAGAGATC-3′,5′-CCGCTCGAGTTATACATCCGCAAACAAC-3′ (open reading frame cloned into NheI/SalI site). Sequence identity of all PCR-amplified fragments was verified by sequencing. For electrophysiological studies AtGAT1 was transferred from pDR196 to pBF1 (29Baukrowitz T. Tucker S.J. Schulte U. Benndorf K. Ruppersberg J.P. Fakler B. EMBO J. 1999; 18: 847-853Crossref PubMed Scopus (78) Google Scholar) using BamHI and ClaI. AtGAT1-GFP and GFP-AtGAT1 were transferred from pUC18-vectors to pBF1 using XmaI and PstI. cRNA was synthesized using the AMBION SP6 mMessage mMachine kit (Ambion, Austin, TX) following the manufacturer's instructions. For quantification of expression, RNA was extracted using a method based on phenol extraction (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.10-7.11Google Scholar) including an additional DNase I treatment. Reverse transcription was performed using the RETROscript kit (Ambion) according to the manufacturer's instructions with oligo(dT) primers and 2 μg of total RNA as template. Relative quantification using real-time PCR was performed on a LightCycler instrument (Roche Diagnostics). The FastStart DNA Master SYBR Green I kit (Roche Diagnostics) was used according to the manufacturer's instructions with MgCl2 at a final concentration of 4 mm and 10 pmol of each primer (AtGAT1: 5′-ATACCTGAAATTCAGGCAACAA-3′, 5′-TAGGTTGTAGGTATACCACAG-3′). Actin (AtAct2) was used as a reference gene (5′-ATTCAGATGCCCAGAAGTCTTGTT-3′, 5′-GAAACATTTTCTGTGAACGATTCCT-3′). AtSag12 was used as a marker for senescence (5′-CGAAGGCGGTTTAATGGATA-3′, 5′-CACCTCCTTCAATTCCAACG-3′). Yeast Growth, Transformation, and Selection—S. cerevisiae strain 22574d (MATα ura3–1, gap1–1, put4–1, uga4–1) (31Jauniaux J.C. Vandenbol M. Vissers S. Broman K. Grenson M. Eur. J. Biochem. 1987; 164: 601-606Crossref PubMed Scopus (107) Google Scholar) was transformed according to Dohmen et al. (32Dohmen R.J. Strasser A.W.M. Höner C.B. Hollenberg C.P. Yeast. 1991; 7: 691-692Crossref PubMed Scopus (319) Google Scholar), and transformants were selected on synthetic dextrose minimal medium (33Burke D. Dawson D. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 172Google Scholar). To test for substrate specificity, transformants were selected on minimal medium supplemented with 20 g/liter of glucose and 1 g/liter of proline, GABA, or citrulline as sole nitrogen source. Transport Assays—Transport assays using S. cerevisiae were done essentially as described previously (34Schwacke R. Grallath S. Breitkreuz K.E. Stransky E. Stransky H. Frommer W.B. Rentsch D. Plant Cell. 1999; 11: 377-392PubMed Google Scholar) using a final concentration of ∼2 × 108 cells/ml, 1.85–55.5 kBq 3H-GABA (Amersham Biosciences) and appropriate amounts of the respective unlabeled GABA. Expression in Xenopus Oocytes—Stage V-VI Xenopus laevis oocytes were injected with 50 ng (50 nl) of AtGAT1 cRNA and were maintained in Barth's medium (88 mm NaCl, 1 mm KCl, 0.33 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 2.4 mm NaHCO3, 10 mm HEPES, pH 7.4, 50 μg/ml of gentamicin, 100 μg/ml of streptomycin, and 100 units/ml of penicillin) at 18 °C for 1–3 days until used in experiments. All of the experiments were performed at 21 ± 1 °C. Experiments were performed in a NaCl buffer containing (in mm): 100 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 PIPES, and 10 HOMOPIPES, pH 7.4. Substrates were added to the buffer solutions as indicated, and the necessary pH adjustments were made. All reagents were purchased from Sigma. Oocytes were voltage clamped by using the Warner Oocyte Clamp (OC-725C; Warner Instrument Corp., Hamden, CT). In the recording experimental chamber, oocytes were initially stabilized in the NaCl buffer, and the composition of the bath was changed as indicated. In all of the experiments, the reference electrodes were connected to the experimental oocyte chamber via agar bridges (3% agar in 3 m KCl). For continuous holding current measurements, the oocyte membrane potential (Vm) was clamped at -50 mV unless otherwise indicated. Currents were low pass filtered at 100 Hz (LPF 8; Warner Instrument Corp.), sampled at 10 Hz (pCLAMP 8.1; Axon Instruments, Union City, CA). Substrate-induced currents were determined by subtracting the base-line current present in NaCl buffer from the evoked current observed after addition of the substrate. The effects of substrate concentration on the steady-state kinetics were determined by non-linear curve fitting of the induced currents (I) to the Michaelis-Menten equation as shown in Equation 1,I=ImaxS×[S]K0.5S+[S](Eq. 1) where S is the substrate, ImaxS is the maximal substrate-induced current, and K0.5S is the substrate concentration at half ImaxS (half-maximal concentration). Curve fittings were performed by using SigmaPlot (SPSS Science, Chicago, IL). Transient Expression in Protoplasts—Transient expression of GFP fusion proteins in tobacco protoplasts was done as described previously (35Dietrich D. Hammes U. Thor K. Suter-Grotemeyer M. Fluckiger R. Slusarenko A.J. Ward J.M. Rentsch D. Plant J. 2004; 40: 488-499Crossref PubMed Scopus (76) Google Scholar), and the samples were examined by using a SP2 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany). Filter settings were 500–520 nm for GFP and 628–768 nm for chlorophyll epifluorescence detection. The Arabidopsis genome contains two members of the ATF amino acid transporter gene family, At1g08230 (AtGAT1) and At5g41800, which exhibit a higher degree of homology to the proline/compatible solute transporters (ProTs) than to members of other subfamilies (Fig. 1) (22Wipf D. Ludewig U. Tegeder M. Rentsch D. Koch W. Frommer W.B. Trends Biochem. Sci. 2002; 27: 139-147Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 36Fischer W.-N. Andre B. Rentsch D. Krolkiewicz S. Tegeder M. Breitkreuz K. Frommer W.B. Trends Plant Sci. 1998; 3: 188-195Abstract Full Text Full Text PDF Scopus (262) Google Scholar). Interestingly, AtGAT1 showed higher homology to a rice protein (OJ1402_H07.15; 62.3% amino acid identity) than to its closest Arabidopsis homolog, At5g41800 (45.8% amino acid identity). Similarly, At5g41800 showed highest homology to a partial mRNA from chickpea (Cicer arietinum AJ004959) and to two rice proteins (OJ1007_H05.2 and P0407B12.25). This is in contrast to members of the ProT subfamily, where proteins from tomato, mangrove, rice, and Arabidopsis display a higher degree of homology within a species than between species. Similar to predictions for other ATF family members, AtGAT1 and At5g41800 encode proteins with a molecular mass of 49.69 and 49.86 kDa, respectively, and 9–12 predicted transmembrane domains (37Schwacke R. Schneider A. Van Der Graaff E. Fischer K. Catoni E. Desimone M. Frommer W.B. Flugge U.I. Kunze R. Plant Physiol. 2003; 131: 16-26Crossref PubMed Scopus (521) Google Scholar). Based on the relatively low sequence identity of AtGAT1 and At5g41800 to ProTs (26–27.2% identity), we wondered whether the newly identified genes code for compatible solute transporters or represent a separate group of amino acid transporters. AtGAT1 Transports GABA with High Affinity—To determine whether AtGAT1 differs in its substrate specificity from members of the ProT gene family, the cDNAs of AtGAT1 (At1g08230) and At5g41800 were isolated by RT-PCR. Subsequently, AtGAT1 and At5g41800 were expressed under the control of the strong PMA1 promoter (vector pDR196) in the S. cerevisiae strain 22574d (28Rentsch D. Laloi M. Rouhara I. Schmelzer E. Delrot S. Frommer W.B. FEBS Lett. 1995; 370: 264-268Crossref PubMed Scopus (277) Google Scholar, 31Jauniaux J.C. Vandenbol M. Vissers S. Broman K. Grenson M. Eur. J. Biochem. 1987; 164: 601-606Crossref PubMed Scopus (107) Google Scholar). The S. cerevisiae mutant 22574d carries mutations in the general amino acid (gap1), proline (put4), and GABA (uga4) permeases and therefore is unable to grow on citrulline, proline, or GABA as the sole nitrogen source. As control, strain 22574d was transformed with the expression vector pDR196 and pDR195 harboring the cDNAs of the proline/compatible solute transporter AtProT2 or of the amino acid permease AtAAP2 (38Kwart M. Hirner B. Hummel S. Frommer W.B. Plant J. 1993; 4: 993-1002Crossref PubMed Scopus (97) Google Scholar, 39Rentsch D. Hirner B. Schmelzer E. Frommer W.B. Plant Cell. 1996; 8: 1437-1446Crossref PubMed Scopus (221) Google Scholar). Growth under selective conditions showed that, like AtProT2, AtGAT1 was able to mediate growth on GABA. However, AtGAT1 could not mediate growth on proline or citrulline (Fig. 2 and data not shown) and histidine was not a substrate for AtGAT1 (data not shown; strain JT16) (40Tanaka J. Fink G.R. Gene. 1985; 38: 205-214Crossref PubMed Scopus (122) Google Scholar). This behavior distinguished AtGAT1 from all transporters of the ATF family characterized so far. In contrast, At5g41800 could not mediate growth on any of the tested substrates. Therefore, At5g41800 was not included in further functional studies. Previous studies described the ProTs as low affinity GABA transporters (Km, 1.7–5 mm) (23Breitkreuz K.E. Shelp B.J. Fischer W.N. Schwacke R. Rentsch D. FEBS Lett. 1999; 450: 280-284Crossref PubMed Scopus (93) Google Scholar, 25Grallath S. Weimar T. Meyer A. Gumy C. Suter-Grotemeyer M. Neuhaus J.M. Rentsch D. Plant Physiol. 2005; 137: 117-126Crossref PubMed Scopus (134) Google Scholar). To examine whether AtGAT1 differed in its kinetic properties, 3H-GABA uptake experiments were performed in 22574d cells expressing AtGAT1. Transport assays showed that AtGAT1 has a much higher affinity for GABA (Km 10 ± 3 μm) than any of the plant transporters characterized before (Fig. 3A). As shown for other transporters of the ATF family, transport rates increased with decreasing pH (Fig. 3B) (39Rentsch D. Hirner B. Schmelzer E. Frommer W.B. Plant Cell. 1996; 8: 1437-1446Crossref PubMed Scopus (221) Google Scholar, 41Fischer W.N. Kwart M. Hummel S. Frommer W.B. J. Biol. Chem. 1995; 270: 16315-16320Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Competition experiments for 3H-GABA uptake in the presence of a 5-fold excess of competitors showed that the GABA-related compounds β-aminobutyric acid and β-alanine reduced GABA transport rates by ∼30%, whereas α-aminobutyric acid did not compete for GABA transport (Fig. 3C). In addition, compounds involved in GABA metabolism (i.e. glutamate, succinic semialdehyde, and succinate) were not competitors for AtGAT1-mediated GABA uptake. Alanine slightly reduced GABA transport rates, whereas histidine as well as compounds that were good competitors for ProT-mediated GABA transport (i.e. d- and l-proline, glycine betaine, and choline) did not reduce GABA uptake activity (23Breitkreuz K.E. Shelp B.J. Fischer W.N. Schwacke R. Rentsch D. FEBS Lett. 1999; 450: 280-284Crossref PubMed Scopus (93) Google Scholar). That none of the substrates tested efficiently competed for GABA uptake suggested that AtGAT1 is a highly selective, high affinity GABA transporter. Electrophysiological Assay of AtGAT1 GABA Transport Kinetics—To determine whether weakly competing compounds were substrates of AtGAT1 and to examine the kinetic properties of AtGAT1 in more detail, substrate-induced currents were analyzed in X. laevis oocytes injected with AtGAT1 cRNA. At a membrane potential of -50 mV and a pH of 5.0, addition of 1 mm GABA to the bathing medium of AtGAT1-expressing oocytes induced inward currents ranging from 96 to 191 nA (Fig. 4B). Current amplitude was dependent on the batch of oocytes and incubation time after cRNA injection. As is commonly observed with many other electrogenic transporters expressed in X. laevis oocytes, the AtGAT1 substrate-evoked current reached a peak followed by slow decay in the presence of the substrate (Fig. 4B) (e.g. Refs. 42Forster I.C. Loo D.D. Eskandari S. Am. J. Physiol. 1999; 276: F644-F649PubMed Google Scholar and 43Whitlow R.D. Sacher A. Loo D.D.F. Nelson N. Eskandari S. J. Biol. Chem. 2003; 278: 17716-17726Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). No GABA-evoked currents were observed in control oocytes (Fig. 4A). Fig. 5A shows the GABA activation curve at a holding potential of -50 mV and an external proton concentration ([H+]out) of 10 μm (pH 5). The apparent affinity of AtGAT1 for GABA was 43 ± 7 μm (n = 3). Likewise, the H+ activation curve at -50 mV and 1 mm GABA was hyperbolic with a K0.5 of 440 ± 40 nm (Fig. 5D). The apparent affinity for GABA (K0.5GABA) appeared not to be voltage dependent in the range from -90 to -10 mV (Fig. 5B), whereas the maximum transport rate (ImaxGABA) increased slightly at depolarized membrane potentials (Fig. 5C). Substrate Selectivity of AtGAT1—Uptake studies such as those shown in Fig. 3C demonstrate whether a compound can alter AtGAT1-mediated GABA transport, presumably by competing for the GABA binding site of AtGAT1. However, these competition studies do not reveal whether a tested compound is in fact a transported substrate of AtGAT1. Thus, to further examine the substrate selectivity of AtGAT1, electrophysiological assays were performed (Fig. 6). GABA, GABA analogs, and other substrates were applied at a concentration of 1 mm, whereas [H+]out was 10 μm and Vm was -50 mV (Fig. 6). An inward current evoked by a substrate was taken as H+-driven, AtGAT1-mediated substrate translocation into the cell. l-Alanine, β-aminobutyric acid, and β-alanine, which only weakly competed for GABA uptake in S. cerevisiae (see Fig. 3C), induced inward currents comparable in magnitude with that induced by GABA. Other GABA-related compounds with longer carbon chains, such as 5-aminovaleric acid, 6-aminocaproic acid, and 8-aminocaprylic acid, were also good substrates. Interestingly, the current induced by butylamine, which lacks the carboxyl group, was similar to that induced by GABA. None of these substrates induced currents in control oocytes. Similar to competition experiments using S. cerevisiae, d- and l-proline as well as glycine betaine and choline were not recognized as substrates (see Figs. 3C and 6). In addition, substrates of the GABA shunt (succinate and glutamate) did not induce currents in AtGAT1-expressing oocytes. Moreover, trigonelline, a betaine present at high concentrations in many legume seeds (44Zheng X.-Q. Hayashibe E. Ashihara H. J. Exp. Bot. 2005; 56: 1615-1623Crossref PubMed Scopus (53) Google Scholar), and the amino acids histidine, glutamine, and norvaline were not transported. The rigid GABA analog nipecotic acid and the amino sulfonate taurine, both substrates of neuronal GABA transporters in mouse (mGAT3 and mGAT4) (2Borden L.A. Neurochem. Int. 1996; 29: 335-356Crossref PubMed Scopus (518) Google Scholar, 45Liu Q.R. Lopez-Corcuera B. Mandiyan S. Nelson H. Nelson N. J. Biol. Chem. 1993; 268: 2106-2112Abstract Full Text PDF PubMed Google Scholar, 46Kragler A. Hofner G. Wanner K.T. Eur. J. Pharmacol. 2005; 519: 43-47Crossref PubMed Scopus (46) Google Scholar), did not induce currents in AtGAT1-expressing oocytes. We reasoned that the compounds that did not compete for GABA uptake in S. cerevisiae but induced currents in AtGAT1-expressing oocytes were low affinity substrates of AtGAT1. Thus, we determined the affinity of AtGAT1 for various substrates. Substrate-induced currents were measured during applications of substrates at concentrations between 5 μm and 100 mm. Currents were plotted against the substrate concentration, and curves were fitted to Equation 1. The apparent affinity of AtGAT1 for individual substrates varied by a factor of 1000 (Table 1). AtGAT1 showed the highest affinity for butylamine with a K0.5 2-fold lower than that for GABA. Moving the amino group closer to the carboxyl group (such as in β-aminobutyric acid and α-aminobutyric acid) progressively reduced the apparent affinity (see Table 1). Increasing the carbon chain backbone from GABA to 8-aminocaprylic acid only marginally affected the K0.5 values (30–80 μm). In addition, 5-aminolevulinic acid was transported with an affinity comparable with that of GABA. Reducing the carbon chain length (e.g. β-alanine) resulted in a much lower substrate affinity (K0.5 200 μm). A similar affinity was