A Novel System A Isoform Mediating Na+/Neutral Amino Acid Cotransport

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 30, Issue of July 28, pp. 22790 –22797, 2000 Printed in U.S.A.

A Novel System A Isoform Mediating Naⴙ/Neutral Amino Acid Cotransport* Received for publication, April 7, 2000, and in revised form May 4, 2000 Published, JBC Papers in Press, May 15, 2000, DOI 10.1074/jbc.M002965200

Dongdong Yao‡, Bryan Mackenzie§, Hong Ming¶, He´le`ne Varoqui¶储, Heming Zhu¶, Matthias A. Hediger§, and Jeffrey D. Erickson‡¶ ** From the ¶Neuroscience Center and the Departments of 储Opthalmology and ‡Pharmacology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112 and the §Membrane Biology Program and Renal Division, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

System A is widely expressed in mammalian cells, where it mediates Na⫹-coupled cellular uptake of small aliphatic amino acids, with alanine, serine, and glutamine being particularly good substrates. It is distinguished from other neutral amino acid transporters like systems L, ASC, and N by the fact that it recognizes N-methylamino acids such as ␣-(methylamino) isobutyric acid (MeAIB),1 does not tolerate substitution of Li⫹ * This work was supported by National Institutes of Health Grant NS36936. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF173682. ** To whom correspondence should be addressed: The Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, 2020 Gravier St., Suite D, New Orleans, LA 70112. Tel.: 504-5990845: Fax: 504-599-0891; E-mail: [email protected]. 1 The abbreviations used are: MeAIB, ␣-(methylamino)isobutyric acid; AAAP, amino acid/auxin permease; EST, expressed sequence tag; GlnT, neuronal glutamine transporter, SAT1, system A transporter 1 (previously GlnT); SAT2, system A transporter 2; SN1/NAT, system N transporter; GABA, ␥-aminobutyric acid; VIAAT/VGAT, vesicular GABA/glycine transporter; TMD, transmembrane domain; DMEM,

for Na⫹, and exhibits trans-inhibition (1–3). An additional important feature of system A is that it is the major amino acid system subject to regulation by environmental conditions, proliferative stimuli, developmental changes, and hormonal signals (4 – 6). Such regulation is thought to be directed at system A, because its substrates play key roles in the overall flux of amino acids between tissues and in delivering energy to amino acid metabolism. In the intestine, system A is localized to basolateral membranes where it plays an important role in the generation of ␣-ketoglutarate, the preferred metabolic fuel of enterocytes over glucose (7). High intracellular alanine levels generated by system A in enterocytes provide the driving force for glutamine uptake through coupled exchange via system L. Glutamine is then converted to glutamate through phosphate-activated glutaminase (PAG), with production of NH4⫹, which exits the basolateral membrane. Aminotransaminase converts glutamate and pyruvate to alanine and ␣-ketoglutarate. Alanine then exits via system L in exchange for additional glutamine. System A, therefore, plays a key role in regulating the availability of essential oxidative metabolites in enterocytes. Alanine is a particularly important nitrogen donor via alanine aminotransferase in peripheral tissues, e.g. the liver and skeletal muscle (8, 9), and in the brain (10). In the liver, system A transports alanine to provide carbon atoms for gluconeogenesis and nitrogen for urea production (8, 9). System A in liver is known to be up-regulated by glucagon and insulin, facilitating the conversion of amino acids to glucose and stimulating urea nitrogen production (11). In the heart and muscle, system A, together with the glutamine transport system N, likely plays an important role in the synthesis of oxidative fuel. The different roles alanine plays in different tissues (8, 9), the different factors (insulin, glucagon, amino acid deprivation, hyperosmotic stress, etc.) that affect the regulation of system A in different tissues (4 – 6), the differences in substrate preferences (e.g. glutamine, proline, glycine) in different tissues (12– 14), and the variants that emerge in transformed cells (15, 16) strongly suggest that a multiplicity of discrete system A isoforms exist. We have recently cloned and functionally identified a neuronal glutamine transporter (GlnT), the first member of the system A family of transporters (17). GlnT was cloned based upon its weak similarity to a family of plasma membrane transporters that are found in plants, yeast, and Caenorhabditis elegans amino acid/auxin permease (AAAP) and to the transporter that mediates uptake of inhibitory amino acid neurotransmitters Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DTT, dithiothreitol; PCR, polymerase chain reaction; kb, kilobase(s); MES, 4-morpholineethanesulfonic acid.

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A cDNA clone encoding a plasma membrane alaninepreferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Naⴙ-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by ␣-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (Km) of 200 –500 ␮M. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to ␣-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.

SAT2: A System A Neutral Amino Acid Transporter

EXPERIMENTAL PROCEDURES

Primary Cultures—Cerebellar primary cultures were prepared from 8-day postnatal (P8) Harlan Sprague-Dawley rats as described (21) with minor modifications. Cerebella were removed from decapitated rat pups, minced, and digested with trypsin (0.25 mg/ml) for 15 min at 37 °C in HEPES-buffered Lebowitz medium containing 0.3% bovine serum albumin. The cell suspension was then placed into Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum (FBS) and centrifuged at 300 ⫻ g for 10 min at 4 °C. This treatment was followed by trituration with a Pasteur pipette in DNase I (50 ␮g/ml) containing DMEM/5% FBS. The cell suspension was allowed to settle for 15 min at room temperature, and the supernatant was transferred to a centrifuge tube on ice. This trituration step was repeated two more times, and the combined supernatants were centrifuged as above. The cell pellet was resuspended in DMEM containing 10% fetal calf serum, 2 mM glutamine, and 100 ␮g/ml penicillin/streptomycin and plated at a density of 15 or 3.75 ⫻ 105 cells (neuronal and astrocytic cultures, respectively) in 65 mm-diameter plastic dishes coated with 10 ␮g/ml polyornithine. The medium for neuronal cultures also contained 25 mM KCl (final concentration). After 18 h, 10 ␮M cytosine arabinoside was added to the neuronal cultures to prevent the replication of non-neuronal cells. The astrocyte culture medium was changed at day 3 in vitro and every other day afterward. The neuronal culture medium was 50% refreshed at day 7. Cloning of SAT2—An oligo(dT)-primed, size-selected cDNA library from cerebellar granule cells (10 days in culture) that was constructed in CDM7/amp, a modified T7 promoter bearing plasmid expression vector, was used (17). Successive subdivisions of the library were screened with a cDNA probe made from a 319-base pair fragment of expressed sequence tag (EST) 184296 amplified by polymerase chain reaction (PCR) using the following two primers, 5⬘-GGCAACTCATATTTCACTATGAAGAGGTAGC-3⬘ and 5⬘-CCAGGTACTACTTCCTTTGGAATGTCAGTA-3⬘ from a human colon cDNA library, gel-purified, and 32P-labeled using [32P]dCTP (NEN Life Science Products) by random priming (Bio-Rad). This fragment corresponds to the conserved putative first membrane-spanning segment of the yeast AAAP homologues (20) and to region I of VIAA/VGAT (18, 19). Southern blots of EcoRI restriction digests of plasmid prepared from overnight cultures were hybridized overnight at 45 °C in a buffer containing 5⫻ SSC; 25% formamide; 5 ⫻ Denhardt’s solution; 50 mM NaPO4 (pH 6.5); 0.1% SDS; 250 ␮g/ml tRNA and the denatured radiolabeled cDNA probe (106cpm/ ml). The filters were washed at 1⫻ SSC; 0.1% SDS at 60 °C. Autoradiographs were analyzed using a BAS2000 phosphor-imaging system (Fuji Biomedical) after 12-h exposure. A 4.2-kilobase (kb) cDNA was obtained and subcloned into pUC18, and overlapping fragments were

sequenced in both directions with the Thermo Sequenase Cycle sequencing kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Preparation of SAT2 Antibody—The coding region for the N-terminal hydrophilic portion of SAT2 (amino acids 1– 65) was amplified by PCR using the primers, 5⬘-CGGGATCCATGAAGACCGAAATGGGAAGGTC-3⬘ and 5⬘-GGAATTCCTAGTCTGTTTCGTACTTCTTCTTCCCCAGAT-3⬘, which include 5⬘-appended BamHI and EcoRI sites (underlined) and an internal stop (italics). This fragment was subcloned into the bacterial expression vector pGEX-KT, and the resulting plasmid was transfected into BL21 cells. Recombinant glutathione S-transferase fusion protein was isolated using a purification module (Amersham Pharmacia Biotech), and then the N terminus of SAT2 was released following thrombin cleavage according to the manufacturer’s instructions. Polyclonal antibodies were produced using the resulting 6-kDa N terminus of rat SAT2 in rabbits by Macromolecular Resources at Colorado State University. Northern Analysis—Poly(A)⫹ RNA was purified from different rat tissues by guanidine isothiocyanate extraction and ultracentrifugation through a cesium trifluoroacetic acid cushion followed by a single round of oligo(dT)-cellulose chromatography (22). 5 ␮g of RNA (A260/280 ⬎ 1.9) was electrophoresed through formaldehyde-agarose gels and stained with ethidium bromide to assure that equivalent amounts of RNA were loaded into each lane. The RNA was then electroblotted onto nylon membranes and hybridized with a oligonucleotide probe (48-mer) against 3⬘ non-coding sequences of SAT2 (bases 1926 –1973) radiolabeled using terminal deoxynucleotidyltransferase (Life Technologies, Inc.) and [32P]dATP (NEN Life Science Products) or random-primed full-length SAT2 cDNA in buffer containing 5⫻ SSC, 50% formamide, 5⫻ Denhardt’s solution, 50 ␮M NaPO4 (pH 6.5), 0.1% SDS, 250 ␮g/ml tRNA for 18 h at 45 °C. The filters were washed in 1⫻ SSC, 0.1% SDS at 60 °C (oligo probe) or 0.1⫻ SSC, 0.1% SDS at 55 °C (cDNA probe) and exposed to Kodak X-Omat film with an intensifying screen at ⫺70 °C. In Situ Hybridization—Sagittal fresh-frozen rat brain sections (15 ␮m) were fixed on slides in 4% formaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature followed by three 5-min washes in PBS. The slides were then immersed in 0.1 M triethanolamine, pH 8, and acetic anhydride (0.25% final) was added under fast stirring. After 10-min incubation, the sections were rinsed in diethylpyrocarbonate-treated water, dehydrated, and delipidated as follows: 70% EtOH for 1 min, 96% EtOH for 1 min, twice 100% EtOH for 2 min, twice chloroform for 5 min, 100% EtOH for 2 min, 96% EtOH for 1 min, and then air-dried. The SAT2 cRNA hybridization probe was prepared as follows: a KpnI-HindIII 3⬘-non-coding fragment of SAT2 (bases 2583–2913) was subcloned into pBluescript KS (Stratagene) and linearized with KpnI and HindIII to generate riboprobes in antisense (T7 polymerase-catalyzed transcription) and sense (T3 polymerase-catalyzed transcription) orientation, respectively. In vitro transcription was performed using 250 ng of linearized plasmid, 62 ␮Ci of [35S]dUTP (NEN Life Science Products), and the Riboprobe Combination System T3/T7 (Promega) according to the manufacturer’s instructions. For hybridization, the sections were covered with hybridization mix containing 80,000 dpm/␮l 35S-labeled cRNA probe in hybridization buffer (50% formamide, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 315 mM NaCl, 10% dextran sulfate, 1⫻ Denhardt’s, 0.1 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA, 0.25 mg/ml yeast total RNA, 10 mM dithiothreitol (DTT), 0.1% sodium thiosulfate, 0.1% sodium dodecyl sulfate) and then coverslipped. After hybridization for 18 h in a humid chamber placed in a 56 °C convection oven, coverslips were removed in 1⫻ SSC/1 mM DTT. Sections were washed twice for 15 min in 1⫻ SSC/1 mM DTT; treated with 20 ␮g/ml RNase A and 1 unit/ml RNase T in 0.5 M NaCl, 10 mM Tris-HCl, pH 8, 1 mM EDTA for 30 min at 37 °C; and then washed successively in 1, 0.5, and 0.2⫻ SSC/1 mM DTT for 15 min each at room temperature and twice in 0.2 ⫻ SSC/1 mM DTT for 20 min at 60 °C. The slides were briefly dipped in water then in 70% ethanol, air-dried, and exposed to Kodak Biomax x-ray film for 96 h. Membrane Preparations and Western Analysis—For primary cultures, cells were rinsed with PBS, harvested by scraping in PBS, centrifuged, and resuspended in PBS containing 1 mM phenylmethylsulfonyl fluoride, 5 mg/l pepstatin, 5 mg/l leupeptin, and 5 mg/l aprotinin, sonicated, and centrifuged at 2000 ⫻ g for 5 min. The resulting supernatants were kept frozen at ⫺80 °C until further use. 5 ␮g of SDSsolubilized cell extracts were size-fractionated under reducing conditions on 8% acrylamide gels and electrophoretically transferred to an nitrocellulose membrane using standard protocols. SAT2, synaptophysin (p38), and glial fibrillary acidic protein were detected using the respective primary antibodies, horseradish peroxidase-conjugated secondary antibodies (Sigma) followed by exposure to film.

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(VIAAT/VGAT) into synaptic vesicles (18 –20). GlnT expression occurs predominantly in nervous tissue and is expressed on the plasma membrane of glutamatergic and some (GABA)ergic (e.g. cerebellar Purkinje cells) neurons. GlnT is a highly efficient glutamine transporter and may be a physiologically important gateway for the precursor of neurotransmitter glutamate via the glutamate/glutamine cycle. Examination of the transport properties of GlnT revealed that not all classical system A substrates (e.g. proline and glycine) could compete with [14C]MeAIB for uptake. In addition, GlnT expression was not found in peripheral organs known to express system A. This indicated that additional members of system A exist and may be expected to express different substrate preferences and display unique tissue specificity. Here, we report the cloning and characterization of SAT2, the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and exhibits broad specificity for neutral amino acids with a preference for alanine. SAT2 is also enriched on glutamatergic neurons in the brain, where it may be an important gateway for alanine uptake to serve, in part, as an amino group donor and alternative precursor of transmitter glutamate. Because alanine plays a key role as a vehicle of nitrogen transport and as an end product of nitrogen catabolism in many tissues, the identification SAT2 will facilitate molecular studies on its functional roles and regulation in various physiological and pathological states.

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SAT2: A System A Neutral Amino Acid Transporter

RESULTS

Cloning and Structural Features of SAT2—Our cloning strategy began with a search of the EST data base for distant mammalian homologues of the vesicular GABA/glycine transporter. A human colon EST was found that displayed moderate sequence identity (25– 45%) within the first putative transmembrane domain (TMD) to a large family of putative amino

acid permeases in the AAAP family of plants, yeast, and C. elegans and in mammals. This EST was homologous to, but differed significantly from, the degenerate oligonucleotide probe used to clone GlnT (17). We screened a rat cDNA library prepared from glutamatergic neuronal cultures with PCR-amplified sequences from this human EST that we obtained from a human colon cDNA library. A cDNA clone with an open reading frame of 1514 bp predicting a highly hydrophobic protein of 504 amino acids and a molecular mass of 55,554 daltons was obtained (Fig. 1). Hydropathy analysis suggests 11 hydrophobic membrane-spanning segments (TMD). The absence of a signal sequence for membrane insertion in the N terminus suggests that it is retained in the cytoplasm leaving the short C terminus located extracellularly. The granule cell cDNA clone has two potential sites for N-linked glycosylation between putative TMDs 5 and 6. Consensus sequences for phosphorylation by protein kinase C exist on the predicted cytoplasmic domain between putative TMDs 6 and 7. Interestingly, SAT2 displays significant homology (approximately 50 –55% identity) to both rat GlnT and to SN1/NAT, a system N transporter (26, 27). Distribution of SAT2—By Northern analysis, the mRNA (⬃4.8 kb) was found to be expressed in most tissues examined, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen, and was absent from testis (Fig. 2). Surprisingly, SAT2 mRNA was present at very low levels in liver, if at all. Equivalent levels of RNA loaded in each well and the lane assignments were confirmed by ethidium staining and by probing with GlnT, a neuron-enriched system A isoform, and with a recently identified homologous isoform specifically enriched in liver (data not shown). In addition, an independent Northern blot was reprobed with SAT2, and no expression in liver was observed. To avoid possible cross-hybridization with homologous family members of system A, Northern blots were probed with a 3⬘-non-coding oligonucleotide as well as with a fulllength cDNA probe washed at high stringency with identical results. Interestingly, the areas containing the most SAT2 mRNA were regions containing neurons (brain and spinal cord) and neuroendocrine cells (adrenal) as well as the intestine and kidney. In situ hybridization of rat brain sections revealed strong labeling in the pyriform cortex, hippocampus, and cerebellar granule layer, regions of the brain that contain neurons that use glutamate as their transmitter (Fig. 3A). Specificity of labeling was confirmed using the sense SAT2 probe (Fig. 3B). In contrast to GlnT, SAT2 mRNA was not observed in cerebellar Purkinje neurons. In situ hybridization of rat spinal cord sections with SAT2 revealed strong labeling of cholinergic motor neurons (data not shown). We also examined the expression of SAT2 in primary cultures of rat cerebellar granule cells, which are a good model for glutamatergic neurons. High levels of expression are observed in neuronal cultures, whereas parallel astrocyte cultures are devoid of immunoreactivity (Fig. 3C). In the neuronal cultures, specific immunoreactivity increases concomitantly with the morphological differentiation of the cells (Fig. 3D). The stimulus-coupled release of glutamate also develops gradually and concomitantly with neuronal differentiation in these cultures (21). Functional Identification of SAT2 in Mammalian Cells—To determine whether SAT2 was a system A transporter like GlnT (17) or a system N transporter like SN1/NAT (26, 27), the cDNA was transiently expressed in fibroblasts using the vaccinia virus/bacteriophage T7 hybrid system and the cultures were incubated with [14C]MeAIB (Fig. 4). The time course of SAT2-specific uptake of [14C]MeAIB (10 ␮M) became saturated by 5 min and remained stable for 20 min. The total uptake of

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Transient Expression and Transport Assay in Mammalian Cells— Monkey kidney fibroblasts (CV-1 cells) were plated on collagen-coated 12-well dishes (2 ⫻ 105 cells/well) in DMEM containing 10% FBS, penicillin (100 units/ml), streptomycin (100 mg/ml), and glutamine (4 mM). Cells were rinsed with DMEM without serum, antibiotics, or glutamine, infected with recombinant T7 vaccinia virus at 10 plaqueforming units/cell for 30 min, and then transfected with plasmid containing SAT2 cDNA in CDM7/amp (1 ␮g/ml) by lipofection in the same medium (23). After 16 h the cells were rinsed with uptake buffer containing 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 1.2 mM CaCl2, 5.6 mM glucose, and 25 mM HEPES, pH 7.4, and preincubated in the same buffer for 5 min at 37 °C. The medium was removed and replaced with fresh buffer at the indicated pH values to which various concentrations of unlabeled alanine or glutamine and 2.5 ␮Ci of [3H]alanine or [3H]glutamine or 0.25 ␮Ci (10 ␮M) of [14C]MeAIB (NEN Life Science Products) was added. For inhibition studies, 1.5 mM unlabeled amino acids were mixed with [14C]MeAIB. Cells were placed in a 37 °C incubator for 2.5 min, and uptake was terminated with a 2.5-ml wash in 4 °C buffer on ice. For Na⫹-free media, NaCl was replaced with equimolar choline Cl or LiCl. For Cl⫺-free media, NaCl was replaced with equimolar NaNO2 or sodium gluconate. Cells were then solubilized in 1 ml of 1% SDS, and radioactivity was measured by scintillation counting in 5 ml of EcoScint (National Diagnostics). Transport measurements were performed in duplicate and repeated at least three times using independent infection/transfections. All experimental conditions with SAT2-transfected cells had corresponding mock-transfected cells in adjacent wells. Functional Characterization of SAT2 in Xenopus Oocytes—Oocytes were isolated from Xenopus laevis (under 2-aminoethylbenzoate anesthesia), treated with collagenase A (Roche Molecular Biochemicals) and stored at 18 °C in modified Barths’ medium (24). SAT2 cDNA was subcloned into the pTLNii vector under SP6, and SAT2 cRNA was synthesized in vitro using the Ambion mMESSAGE mMACHINE 228 kit. Oocytes were injected with 50 ng of SAT2 cRNA and incubated 2 days before performing radiotracer or voltage-clamp experiments at 22 °C. Standard Na⫹ uptake medium comprised 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES (pH 7.5 with Tris base). For Na⫹-free or low Na⫹ media, NaCl was replaced by equimolar choline chloride. For pH sensitivity experiments, Na⫹ media were buffered at pH 6.5– 8.6 using 0 –5 mM MES, 0 –5 mM HEPES, and 0 –5 mM Tris base. A two-microelectrode voltage clamp was used to measure currents in control (water-injected) oocytes and oocytes injected with SAT2 cRNA. Microelectrodes (resistance, 0.5–3 M⍀) were filled with 3 M KCl. Oocytes were clamped at a holding potential (Vh) of ⫺50 mV and step-changes in membrane potential (Vm) applied (from ⫹50 to ⫺150 mV in 20-mV increments), each for a duration of 100 ms, before and after the addition of substrate; current was low-pass-filtered at 500 Hz and digitized at 5 kHz. Test solutions were washed out with substrate-free medium (100 mM choline chloride) at pH 7.5 for several minutes. Steady-state data (obtained by averaging the points over the final 16.7 ms at each Vm step) were fitted to Eq. 11.3 of Mackenzie (24), for which I is the evoked current (i.e. the difference in steady-state current measured in the presence and absence of substrate), Imax the derived current maximum, S S the concentration of substrate S (Na⫹ or amino acid), K0.5 the substrate concentration at which current was half-maximal, and nH the Hill coefficient for S. Additionally, continuous current monitoring was performed at Vh ⫽ ⫺70 mV, for which the current was low-pass-filtered at 20 Hz (sampling at 1 Hz). Na⫹/L-alanine coupling stoichiometry was determined by direct comparison of net inward charge with [3H]alanine accumulation in individual oocytes under voltage clamp (24, 25). Oocytes were clamped at Vh ⫽ ⫺70 mV and superfused with standard Na⫹ medium (pH 7.5) plus 500 ␮M L-[3H]alanine (NEN Life Science Products; final specific activity, 1–2 MBq䡠mmol⫺1) for 5 min before washing out with Na⫹ medium. The alanine-evoked current was integrated with time to obtain the alaninedependent charge (QAla) and converted to a molar equivalent using the Faraday conversion. Oocytes were solubilized with 10% SDS, and the 3 H content was measured by liquid scintillation counting.

SAT2: A System A Neutral Amino Acid Transporter

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FIG. 2. Widespread expression of SAT2 mRNA. Northern analysis of 5 ␮g of oligo(dT)-purified poly(A)⫹ RNA shows an approximate 4.8-kb transcript in most tissues with the highest levels seen in the brain, spinal cord, adrenal, kidney, and colon; lower levels in muscle, heart, fat, stomach, and spleen; and none detectable in liver or testis. The position of 28S and 18S ribosomal bands (in kilobases) are shown on the left.

[14C]MeAIB was approximately 5-fold greater in the transfected cells (29 pmol/well) than in the mock-transfected cells (6 pmol/well) at 2.5 min of incubation. Kinetic analysis of MeAIB uptake by SAT2 at pH 7.4 was saturable, with a Michaelis constant (Km) of 530 ⫾ 53 ␮M; n ⫽ 9 (Fig. 4A). The transporter encoded by SAT2 has an absolute requirement for extracellular Na⫹ ions, because choline and lithium cannot effectively substitute for it in transfected cells (Fig. 4B). Replacement of chloride with nitrate or acetate resulted in uptake changes of less than 30%, suggesting that anions may not be thermodynamically coupled to [14C]MeAIB uptake. A characteristic feature of SAT2 is its pH sensitivity. [14C]MeAIB uptake mediated

by SAT2 is strongly pH-sensitive with uptake at pH 8.2 being approximately six times greater than that seen at pH 6.6 (Fig. 4C). To further define the characteristics of SAT2 we tested the ability of various amino acids (1.5 mM) to compete with [14C]MeAIB (10 ␮M) for uptake (Fig. 4D). Alanine is the preferred substrate for SAT2. Serine, proline, and methionine are also effective [14C]MeAIB uptake competitors. Asparagine, glutamine, glycine, and histidine are less effective inhibitors, whereas arginine, ␤-alanine, glutamic acid, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, and valine are ineffective inhibitors. MeAIB inhibition of [14C]MeAIB uptake was approximately 80% at 1.5 mM concentration. A direct comparison of alanine and glutamine transport by SAT2 was also determined (Fig. 4E). Kinetic analysis indicate that the affinity of alanine for SAT2 was similar to MeAIB (Km ⫽ 529 ⫾ 50 ␮M; Vmax ⫽ 656 ⫾ 35 nmol/min/well; n ⫽ 4) but significantly greater than for glutamine (Km ⫽ 1.65 ⫾ 0.27 mM; Vmax ⫽ 389 ⫾ 40 nmol/min/well; n ⫽ 5). Functional Characterization of SAT2 in Xenopus Oocytes— The functional characteristics of SAT2 were also explored by applying radiotracer and voltage-clamp techniques in Xenopus oocytes expressing SAT2. SAT2 mediates saturable, Na⫹-dependent amino acid transport in a rheogenic manner. In the presence of Na⫹, although L-alanine evoked currents of ⬍⫺3 nA in control oocytes (see also Ref. 28), L-alanine evoked reversible inward currents of up to ⫺1000 nA in oocytes expressing SAT2 (Fig. 5A). Smaller currents were obtained in response to L-glutamine, and the current evoked by MeAIB was only about 25% of the L-alanine-evoked current. SAT2 stimulated

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FIG. 1. Primary amino acid sequence and proposed structure of SAT2. Eleven putative transmembrane domains, potential sites for N-linked glycosylation (three-pronged branches) and phosphorylation by protein kinase C (P inside the circle) are indicated. White balls (with black letters) represent conserved residues between SAT1 and SAT2. Black balls (with white letters) represent amino acids unique to SAT2. The cytoplasm is shown above and the extracellular space below; ⫺, represents acidic residues; ⫹, basic residues.

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3 ⫺1 L-[ H]alanine (500 ␮M) uptake 25-fold (61 ⫾ 16 pmol䡠min , mean ⫾ S.E.) over control oocytes (2.4 ⫾ 0.5 pmol䡠min⫺1) and [14C]MeAIB uptake (200 ␮M) 5-fold to 0.9 ⫾ 0.07 pmol䡠min⫺1 (n ⫽ 6 –12 oocytes in each group). MeAIB evokes a tiny outward current in native oocytes (28) and accelerates endogenous ala-

FIG. 4. Functional identification of SAT2 as a system A transporter. A, kinetic analysis of uptake of MeAIB by SAT2 in transiently transfected CV-1 cells. Saturation isotherm of initial velocity (2.5 min) of MeAIB (0.1–3.2 mM) uptake by SAT2. Inset, Lineweaver-Burk analysis of SAT2 initial uptake velocity. B, MeAIB uptake by SAT2 is Na⫹-dependent and Li⫹-intolerant. Transport of [14C]MeAIB (10 ␮M) was measured at 2.5 min in NaCl-containing buffer (control, 100%), Na⫹-deprived buffer (replaced by Li⫹ or choline), or Cl⫺-deprived buffer (replaced by gluconate or NO2). Error bars represent the standard error of the mean (n ⫽ 3–9). C, MeAIB uptake by SAT2 is strongly pH-sensitive. [14C]MeAIB (10 ␮M) uptake was terminated after 2.5 min. Transport observed in SAT2-expressing cells (filled squares) is greatly stimulated by a decrease in extracellular H⫹ ion concentration. Background transport in mock-transfected cells (open circles) is only moderately affected by pH. D, alanine is the preferred substrate for SAT2. Transport of [14C]MeAIB (10 ␮M) was measured at 2.5 min in the absence (Control, 100%) or presence (normalized results) of various amino acids (1.5 mM). All amino acids are of the L-form. E, saturation analysis of initial velocity (2.5 min) alanine (crosses) and glutamine (filled triangles) uptake by SAT2.

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FIG. 3. SAT2 is expressed in glutamatergic neurons in the brain. A, in situ hybridization of rat brain sagittal sections with 35Slabeled cRNA probes for SAT2 (left, antisense; right, sense) reveal enriched signals for SAT2 in areas of the brain containing glutamatergic cell populations. Cx, cortex, Cpu, caudate-putamen; Hc, hippocampus; Th, thalamus; Cb, cerebellum. B, specificity of the SAT2 antibody. Inclusion (⫹) of the GST-SAT2 fusion protein (5 ␮g/ml) abolishes immunostaining of the 55-kDa band only. C, SAT2 is detected in cerebellar granule cells (n) and not astrocyte cultures. (a). The same samples were also blotted and probed for synaptophysin (p38) and glial fibrillary acidic protein, markers of neurons, and glia, respectively. D, SAT2 expression is developmentally up-regulated in postnatal cultures (postnatal days 8 –18) of cerebellar granule cells and reaches maximal levels by day 7 (about postnatal day 15).

nine transport (29), so that a phenomenon specific to the oocyte expression system may, in part, account for why MeAIB fluxes and currents were significantly smaller than those for L-alanine despite the similarity of the Km values for these substrates observed in transfected CV-1 cells. The substrate selectivity of SAT expressed in oocytes (data not shown) reflected the pattern observed using the T7 vaccinia system, and SAT2 was stereospecific for the L-isomer, at least in the case of alanine. In oocytes expressing SAT2, the L-alanine-evoked currents (at saturating amino acid) showed a curvilinear dependence on membrane potential (Vm) (Fig. 5B). The current/voltage relationship was roughly linear between ⫺150 and ⫺30 mV, and no reversal of the currents was observed up to ⫹50 mV. The L-alanine-evoked currents followed Michaelis-Menten saturaAla tion kinetics. At ⫺70 mV, K0.5 (L-alanine concentration at which currents were half-maximal) was around 200 ␮M (Fig. Ala 5C) with a Hill coefficient (nH) for alanine of ⫺1. K0.5 was largely independent of Vm, except at depolarized potentials (more positive than ⫺10 mV), rising to 1 mM at ⫹30 mV. The current-voltage relationships for subsaturating (200 ␮M) ⫹ L-alanine were determined as a function of extracellular Na concentration (Fig. 5F). At any given Vm, the currents were larger at higher [Na⫹]o, indicating that L-alanine transport mediated by SAT2 is driven by the Na⫹ electrochemical gradient. The saturation kinetics for Na⫹ were determined as a Ala function of Vm at 0.2 mM L-alanine. At ⫺70 mV, K0.5 was 20 mM and the Hill coefficient (nH) for Na⫹ was ⫺1 (Fig. 5G). nH for Ala Na⫹ did not vary with Vm, however, K0.5 exhibited significant voltage dependence, rising from ⬍10 mM at hyperpolarized Vm to almost 50 mM at ⫹50 mV (Fig. 5H). These data suggest that only one Na⫹ binds to SAT2 (or that there is no cooperativity between the binding of multiple Na⫹) and that Na⫹ binding is voltage-sensitive. The Na⫹/L-alanine coupling stoichiometry was directly determined by comparing the alanine-dependent charge QAla (converted to a molar equivalent assuming monovalence) with

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the concomitant accumulation of L-[3H]alanine in individual voltage-clamped oocytes at ⫺70 mV (Fig. 5I). QAla correlated with 3H accumulation in a linear fashion (with a slope of 1.04), justifying our use of the amino acid-evoked current as a direct index of amino acid transport. The Na⫹/L-alanine coupling coefficient (n) was 1.19 ⫾ 0.09, and the mean QAla was not significantly different from the 3H accumulation in a paired t test, indicating that Na⫹-coupled, L-alanine transport in SAT2 proceeds with 1:1 stoichiometry. Unlike the Na⫹/glucose cotransporter SGLT1 (24), there was no appreciable slippage (uncoupled current or uniport) of Na⫹, because the shift in inward current as a result of switching from 0 to 100 mM Na⫹ in oocytes expressing SAT2 (Fig. 5A) was not significantly different from the current observed in control oocytes. DISCUSSION

We have cloned and functionally identified SAT2, the second member of the system A family of neutral amino acid transporters. SAT2 shares 55% identity with GlnT (17), which we

renamed SAT1. In addition, SAT2 shares 52% identity with SN1/NAT, a recently identified system N transporter expressed predominantly in liver but also found in brain and kidney (26, 27). The functional properties of the transport activity of SAT2 observed in transfected mammalian cells indicate that it is alanine-preferring, capable of transporting MeAIB and small aliphatic amino acids, and intolerant of Li⫹ substitution for Na⫹. These characteristics are discriminating features of system A and system N in tissue-cultured cells. Additional properties of SAT2 revealed in this study include voltage dependence (1:1 Na⫹ stoichiometry) and pH sensitivity consistent with previously described properties of system A (1– 6). Although both systems A and N exhibit Na⫹ dependence and are pHsensitive, SN1/NAT has been identified as a proton antiporter and appears to behave as a glutamine-gated Na⫹/H⫹ exchanger (26). Thus, changes in intra- or extracellular pH will affect the direction of flux through SN1/NAT. Changes in extracellular pH significantly increase the efficiency of both SAT1

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FIG. 5. Currents associated with SAT2 expressed in Xenopus oocytes. A, typical continuous current recordings from a single oocyte injected with SAT2 cRNA and voltage-clamped at ⫺70 mV. The oocyte was superfused with 100 mM NaCl medium at pH 7.5 (hatched boxes), and 10 mM amino acids as indicated were applied for the periods shown by the black boxes before washing out with choline chloride medium (blank boxes). B–H, saturation kinetics and voltage dependence of the Na⫹-dependent L-alanine currents in SAT2. All data presented (B–H) were derived in a single oocyte expressing SAT2, and errors represent the error in the estimate of kinetic parameters according to Eq. 11.3 of Ref. 24. B, current/voltage relationship for 10 mM L-alanine in 100 mM NaCl. C, D, and E, saturation kinetics of L-alanine-evoked currents were determined over the Vm range ⫺150 to ⫹50 mV by applying L-alanine at 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, and 10 mM in 100 mM NaCl. At Vm ⫽ ⫺70 mV Ala Ala (C), the derived kinetic parameters were K0.5 ⫽ 0.19 ⫾ 0.03 mM, Imax ⫽ ⫺284 ⫾ 11 nA, and nH for L-alanine was 0.9 ⫾ 0.1 (r2 ⫽ 0.99). The Hill Ala Ala coefficient (nH) for L-alanine did not differ significantly at other Vm (data not shown). The voltage dependence of K0.5 (D) and Imax (E) are also presented. F, I/V relationships for 0.2 mM L-alanine as a function of Na⫹ concentration. G and H, Na⫹ saturation kinetics were determined from the currents evoked by subsaturating (0.2 mM) L-alanine at 4, 5, 10, 20, 30, 50, 60, 80, and 100 mM NaCl. At Vm ⫽ ⫺70 mV (G), the derived kinetic Na Na parameters were K0.5 ⫽ 20.2 ⫾ 1.5 mM, Imax ⫽ ⫺96 ⫾ 3 nA, and nH for L-alanine was 1.1 ⫾ 0.1 (r2 ⫽ 1.0). The Hill coefficient for Na⫹ did not Na significantly vary with Vm (data not shown). The relationship of K0.5 to Vm is shown in H. I, Na⫹/L-alanine coupling coefficient determined by 3 3 L-[ H]alanine uptake under voltage clamp. Oocytes were clamped at ⫺70 mV, and 500 ␮M L-[ H]alanine was applied for 5 min. The alanine-dependent charge (QAla) was compared with concomitant tracer accumulation in the same oocytes expressing SAT (filled circles). Charge and tracer accumulation in control oocytes (mean ⫾ S.E., bidirectional, n ⫽ 3 oocytes; open circle) was first subtracted. Linear regression of SAT data (r2 ⫽ 0.43) gave a slope of 1.04. The ratio of QAla:3H accumulation was 1.19 ⫾ 0.09 (mean ⫾ S.E., n ⫽ 6 oocytes), and there was no significant difference between QAla and 3H accumulation (inset) according to Student’s paired t test (t ⫽ 1.9).

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SAT2: A System A Neutral Amino Acid Transporter mitochondrial enzyme (46 – 49), for the synthesis of the neurotransmitter pool. A second major metabolic fate of glutamate once accumulated by astrocytes is a reaction with pyruvate to form alanine via the alanine aminotransferase pathway (50). ␣-Ketoglutarate together with alanine (an amino group donor) have been shown to be efficiently converted to transmitter glutamate via an aminooxyacetic acid-inhibitable transamination reaction and can maintain the release of glutamate in cerebellar granule cells and in brain slices (51, 52). The major synthesis of the tricarboxylic acid intermediates such as ␣-ketoglutarate also occurs in astrocytes, because only they possess pyruvate carboxylase and are able to perform carbon dioxide fixation (53–55). Thus, neuronal ␣-ketoglutarate and alanine, in addition to glutamine, are supplied by astrocytes (56 –58) and are actively accumulated into neurons (10, 14, 50, 59 – 62). The astrocyte-to-neuron metabolic shuttle via SN1/NAT, SAT1, and SAT2 may enable not only replenishment of neurotransmitter glutamate (and GABA) but also may provide neurons, in general, with the capacity for oxidative metabolism and ATP generation. REFERENCES 1. Barker, G. A., and Ellory, J. C. (1990) Exp. Physiol. 75, 3–26 2. Christensen, H. N. (1990) Physiol. Rev. 70, 43–77 3. Palacin, M., Estevez, R., Bertran, J., and Zorzano, A. (1998) Physiol. Rev. 78, 969 –1054 4. Guidotti, G., Borghetti, A. F., and Gazzola, G. C. (1978) Biochim. Biophys. Acta 515, 329 –366 5. Haussinger, D., Lang, F., and Kilberg, M. S. (1992) in Mammalian Amino Acid Transport. Mechanisms and Control (Kilberg, M. S., and Haussinger, D., eds) pp. 113–130, Plenum Press, New York 6. McGivan, J. D., and Pastor-Anglada, M. (1994) Biochem. J. 299, 321–334 7. Bradford, N. M., and McGivan, J. D. (1982) Biochim. Biophys. Acta 689, 55– 62 8. Felig, P. (1975) Annu. Rev. Biochem. 44, 933–955 9. Christensen, H. N. (1982) Physiol. Rev. 62, 1193–1233 10. Westergaard, N., Varming, T., Peng, L., Sonnewald, U., Hertz, L., and Schousboe, A. (1993) J. Neurosci. Res. 35, 540 –545 11. Christensen, H. N., and Kilberg, M. S. (1995) Nutr. Rev. 53, 74 –76 12. Kilberg, M. S., Handlogten, M. E., and Christensen, H. N. (1980) J. Biol. Chem. 255, 4011– 4019. 13. Ardawi, M. S. (1986) Biochem. J. 238, 131–135 14. Su, T.-Z., Campbell, G. W., and Oxender, D. L. (1997) Brain Res. 757, 69 –78 15. Dudeck, K. L., Dudenhausen, E. E., Chiles, T. C., Fafournoux, P., and Kilberg, M. S. (1987) J. Biol. Chem. 262, 12565–12569 16. Lin, G., McCormick, J. E., and Johnstone, R. M. (1994) Arch. Biochem. Biophys. 312, 308 –315 17. Varoqui, H., Zhu, H., Yao, D., Ming, H., and Erickson, J. D. (2000) J. Biol. Chem. 275, 4049 – 4054 18. McIntire, S. L., Reimer, R. J., Schuske, K., Edwards, R. H., and Jorgensen, E. M. (1997) Nature 389, 870 – 876 19. Sagne, C., El Mestikawy, S., Isambert, M. F., Hamon, M., Henry, J. P., Giros, B., and Gasnier, B. (1997) FEBS Lett. 417, 177–183 20. Young, G. B., Jack, D. L., Smith, D. W., and Saier, M. H., Jr. (1999) Biochim. Biophys. Acta 1415, 306 –322 21. Gallo, V., Ciotti, M. T., Coletti, A., Aloisi, F., and Levi, G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7919 –7923 22. Yokota, R., and Arai, K. (1987) Methods Enzymol. 154, 3–28 23. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8122– 8126 24. Mackenzie, B. (1999) in Biomembrane Transport (Van Winkle, L. J., ed) pp. 327–342, Academic Press, San Diego, CA 25. Mackenzie, B., Loo, D. D. F., and Wright, E. M. (1998) J. Membr. Biol. 162, 101–106 26. Chaudhry, F. A., Reimer, R. J., Krizaj, D., Barber, D., Storm-Mathisen, J., Copenhagen, D. R., and Edwards, R. H. (1999) Cell 99, 769 –780 27. Gu, S., Roderick, H. L., Camacho, P., and Jiang, J. X. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3230 –3235 28. Mackenzie, B., Harper, A. A., Taylor, P. M., and Rennie, M. J. (1994) Pflu¨gers Arch. 426, 121–128 29. Palacin, M., Werner, A., Dittmer, J., Murer, H., and Biber, J. (1990) Biochem. J. 270, 189 –195 30. Hediger, M. A. (1999) Am. J. Physiol. 277, F487–F492 31. Sugawara, M., Nakanishi, T., Fei, Y.-J., Huang, W., Ganapathy, M. E., Leibach, F. H., and Ganapathy, V. (2000) J. Biol. Chem. 275, 16473–16477 32. Hertz, L. (1979) Prog. Neurobiol. 13, 277–323 33. Laake, J. H., Takum, Y., Eide, J., Torgner, I. A., Roberg, B., Kvamme, E., and Ottersen, O. P. (1999) Neuroscience 88, 1137–1151 34. Bradford, H. F., Ward, H. K., and Thomas, A. J. (1978) J. Neurochem. 30, 1453–1459 35. Hamberger, A. C., Han Chiang, G., Nyle´n, E. S., Scheff, S. W., and Cotman, C. W. (1979) Brain Res. 168, 513–530 36. Hamberger, A. C., Han Chiang, G., Sandoval, E., and Cotman, C. W. (1979) Brain Res. 168, 531–541 37. Shank, R. P., and Aprison, M. H. (1981) Life Sci. 28, 837– 842 38. Erecinska, M., and Silver, I. A. (1990) Prog. Neurobiol. 35, 245–296

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and SAT2 function by affecting the Vmax of transport. However, the direct measurement of the 1:1 stoichiometry of Na⫹:alanine transport by SAT2 observed in voltage-clamped Xenopus oocytes argues against protons being involved in the thermodynamic coupling of system A-mediated transport. Both SAT1 and SAT2 are capable of transporting MeAIB, yet they exhibit differences with respect to their substrate selectivity and their distribution in various tissues. Although both recognize alanine as a substrate, SAT1 is a more efficient glutamine transporter than SAT2. The efficiency (Vmax/Km) of alanine transport by SAT2 is approximately four times greater than that of glutamine. SAT2 effectively recognizes proline compared with SAT1. SAT1 is expressed predominantly in brain and spinal cord and is absent from peripheral tissues like heart, muscle, kidney, and lung where SAT2 mRNA is present. The expression of SAT2 in the intestine is consistent with its role in regulating the availability of oxidative metabolites. Preliminary in situ hybridization data suggest the mRNA signal in kidney resides in the medulla rather than in cortex (data not shown), but the precise localization of SAT2 in kidney has not yet been determined. It is possible that it transports amino acids as osmolytes into renal cells in response to hyperosmotic stress (30). SAT2 mRNA is not observed in liver, a tissue where both insulin and glucagon stimulate system A activity to facilitate the conversion of amino acids to glucose and to stimulate urea nitrogen production. A related article on this subject reports on the sequence ATA2 that is identical to SAT2, with a Northern blot showing dramatic expression in the liver (31). This discrepancy in tissue distribution may be related to the higher stringency conditions used for Northern analysis in the present study. The existence of additional distinct system A isoforms has been suggested in the literature (15, 16), and the unique role system A plays in liver is consistent with the concept that distinct isoforms exist to allow appropriate regulation, for example in response to a high protein diet or glucose deficiency. Alternatively, SAT2 expression in hepatocytes might be tightly regulated and intimately dependent upon the metabolic/functional disposition of the animal. SAT2 mRNA is enriched in glutamatergic neurons in the brain, is selectively expressed in neuronal-rich cultures and not in astrocyte-rich cultures, and is up-regulated during neuronal differentiation. These results suggest that SAT2 may also play an important role in glutamatergic function in addition to SAT1. Although glutamine is the preferred precursor for neurotransmitter glutamate, it cannot be the only source because the amount of glutamate released exceeds the amount of glutamine that enters glutamatergic neurons (32). In addition, PAG is present at much lower levels in some glutamatergic pathways than in others (33), further suggesting that alternative sources for transmitter glutamate must exist. It is well recognized that glutamatergic neurons depend upon glia for maintenance of their neurotransmitter pool (34 – 42). After release from neurons during neurotransmission, glutamate is rapidly cleared from the synaptic region by glutamate transporters that are expressed predominantly on astrocytes (43, 44). There, glutamate is partly converted to glutamine by the astrocyte-specific enzyme glutamine synthetase (45). Recently, a system N transporter (SN1/NAT) that acts as a Na⫹/H⫹gated glutamine effluxer was identified and is restricted to glial cells in the rat brain (26, 27). SAT1, a glutamine-preferring system A transporter, is expressed on glutamatergic neurons and is absent from astrocytes (17). The identification of systems A and N in the brain supports the notion of the glutamate/glutamine cycle in the recycling of transmitter glutamate. Thus, glutamine may exit astrocytes via SN1/NAT and enter glutamate neurons via SAT1 to be made available to PAG, a

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A Novel System A Isoform Mediating Na+/Neutral Amino Acid Cotransport Dongdong Yao, Bryan Mackenzie, Hong Ming, Hélène Varoqui, Heming Zhu, Matthias A. Hediger and Jeffrey D. Erickson J. Biol. Chem. 2000, 275:22790-22797. doi: 10.1074/jbc.M002965200 originally published online May 15, 2000

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