SNAT2 Amino Acid Transporter Is Regulated by Amino Acids of the SLC6

14 Pages·2009·0.82 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.284,NO.17,pp.11224–11236,April24,2009
©2009byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
SNAT2 Amino Acid Transporter Is Regulated by Amino Acids
(cid:1)
of the SLC6 -Aminobutyric Acid Transporter Subfamily in
Neocortical Neurons and May Play No Role in Delivering
Glutamine for Glutamatergic Transmission*
Receivedforpublication,August20,2008,andinrevisedform,February6,2009Published,JBCPapersinPress,February24,2009,DOI10.1074/jbc.M806470200
SukhjeevanGrewal‡1,NorahDefamie‡1,XiongZhang‡,Ste´phanieDeGois‡,AliShawki§,BryanMackenzie§,
ChuChen‡,He´le`neVaroqui‡,andJeffreyD.Erickson‡2
Fromthe‡NeuroscienceCenter,LouisianaStateUniversityHealthScienceCenter,NewOrleans,Louisiana70112andthe
§DepartmentofMolecularandCellularPhysiology,UniversityofCincinnatiCollegeofMedicine,Cincinnati,Ohio45267-0576
SystemAtransportersSNAT1andSNAT2mediateuptakeof Centralneuronsexpressthesodium-coupledneutralamino
neutral(cid:2)-aminoacids(e.g.glutamine,alanine,andproline)and acidtransportersSNAT1andSNAT2(alsoknownasSLC38A1,
areexpressedincentralneurons.Wetestedthehypothesisthat GlnT,SAT1,orATA1andSLC38A2,SAT2,orATA2,respec-
SNAT2 is required to support neurotransmitter glutamate tively) (1–10), two of the three SLC38 gene family members
synthesisbyexaminingspontaneousexcitatoryactivityafter (reviewedinRef.11)thatcollectivelyaccountfortheSystemA
inducingorrepressingSNAT2expressionforprolongedperi- aminoacidtransportactivityclassicallydescribedinmostcell
ods.WestimulateddenovosynthesisofSNAT2mRNAand types(12,13).SystemAcatalyzestheunidirectionaluptakeof
increased SNAT2 mRNA stability and total SNAT2 protein smallaliphaticneutralaminoacids,especiallyalanine,cysteine,
andfunctionalactivity,whereasSNAT1expressionwasunaf- glutamine, glycine, methionine, proline, and serine. Among
fected. Increased endogenous SNAT2 expression did not these, glutamine is around 10-fold more abundant than any
affect spontaneous excitatory action-potential frequency otheraminoacidinextraneuronalspace(14–16),which,com-
over control. Long term glutamine exposure strongly binedwiththehighaffinityofSystemAtransportersforgluta-
repressedSNAT2expressionbutincreasedexcitatoryaction- mine(Km(cid:1)0.2–0.5mM),makesglutaminethepredominant
potential frequency. Quantal size was not altered following substrateforSystemAinneuronsofthebrain.Glutamine,in
SNAT2 induction or repression. These results suggest that additiontoglucose,isamajorprecursorforglutamate(17),and
spontaneous glutamatergic transmission in pyramidal neu- manyneuronalcellbodiesinthebrainexpressphosphate-acti-
ronsdoesnotrelyonSNAT2.Tooursurprise,repressionof vatedglutaminase(18,19)andcontainhighlevelsofglutamate.
SNAT2activitywasnotlimitedtoSystemAsubstrates.Tau- WhereasSystemNtransportersmayservetheexodusofgluta-
rine, (cid:1)-aminobutyric acid, and (cid:3)-alanine (substrates of the minefromtheastrocyte(9,20,21),theseobservationspresent
SLC6 (cid:1)-aminobutyric acid transporter family) repressed thepossibilitythatSNAT1andSNAT2playaroleinproviding
SNAT2 expression more potently (10(cid:4)) than did System A central neurons with the glutamine required for synthesis of
transmitter glutamate (22), but the evidence is controversial
substrates; however, the responses to System A substrates
(23).
weremorerapid.SinceATF4(activatingtranscriptionfactor
HerewetestedthehypothesisthatSNAT2providesneurons
4)andCCAAT/enhancer-bindingproteinareknowntobind
with glutamine required for neurotransmitter glutamate syn-
to an amino acid response element within the SNAT2 pro-
thesisbymeasuringspontaneousglutamatergicactivityinpri-
moter and mediate induction of SNAT2 in peripheral cell
mary neuronal cultures in which we altered endogenous
lines, we tested whether either factor was similarly induced
SNAT2expressionlevels.AdaptiveregulationofSystemA(up-
byaminoaciddeprivationinneurons.Wefoundthatgluta-
regulation in amino acid-deprived ((cid:2)AA)3 conditions and
mineandtaurinerepressedtheinductionofbothtranscrip-
repression of System A activity when substrates are reintro-
tionfactors.OurdatarevealedthatSNAT2expressioniscon-
duced)hasbeenwidelystudiedinperipheralcelltypes(12,13).
stitutivelylowinneuronsunderphysiologicalconditionsbut
Suchconditionshavenoeffectonglutaminetransportactivi-
potently induced, together with the taurine transporter
tiesattributedtoSystemsL,N,orASC(24).Innonneuronalcell
TauT,inresponsetodepletionofneutralaminoacids.
types in which only one System A isoform is expressed,
increasedfunctionalactivityresultsfromdenovosynthesisof
SNAT2mRNAandproteininresponseto(cid:2)AAtreatment(25–
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth
Grants1P29RR16816(toH.V.)andNS36936(toJ.D.E.).Thisworkwasalso
supportedbytheUniversityofCincinnati(toB.M.). 3Theabbreviationsusedare:(cid:2)AA,aminoaciddeprivation;AARE,aminoacid
1Bothauthorscontributedequallytothiswork. response element; ACD, actinomycin D; (cid:1)-AIB, (cid:1)-aminoisobutyric acid;
2Towhomcorrespondenceshouldbeaddressed:NeuroscienceCenter,Lou- C/EBP(cid:1), CCAAT/enhancer-binding protein; CHX, cycloheximide; GABA,
isianaStateUniversityHealthSciencesCenter,2020GravierSt.,Ste.D, (cid:2)-aminobutyricacid;MeAIB,(cid:3)-methylaminoisobutyricacid;mEPSC,mini-
New Orleans, LA 70112. Tel.: [phone]; Fax: [phone]; E-mail: atureexcitatorypostsynapticcurrent;PBS,phosphate-bufferedsaline;RT,
[email protected]. reversetranscription.
11224 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER17•APRIL24,2009
This is an Open Access article under the CC BY license.
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
27). Adaptive regulation of System A was considered to be culture medium with B27 is (cid:4)289 mosM, and that of Krebs-
restrictedtotheeffectsofitsownsubstrates,sincetheaddition Ringer bicarbonate buffer used for (cid:2)AA treatment is 300
ofSystemAsubstratesblockstheincreaseintransportactivity mosM.
observedinresponseto(cid:2)AAtreatment,whereasotheramino (cid:2)AA of Cultured Neuronal Cells—Regulation studies were
acids(leucine,theclassicSystemLinhibitor2-aminobicyclo- conducted in differentiating neuronal cultures (12 days in
(2,2,1)-heptane-2-carboxylicacid,anionic(glutamate),andcat- vitro),atthetimeoffunctionalexcitatorysynapsematuration
ionic(arginine)aminoacids)producenosucheffect(24–31). andmajoronsetofregulatedglutamateexocytosisviaVGLUT1
At the genomic level, an intronic amino acid response ele- (vesicularglutamatetransporterisoform1(SLC17A7,BNPI))
ment (AARE) that is responsive to adaptive regulation and (42). To treat the cells with (cid:2)AA medium, the Neurobasal
binds ATF4 (activating transcription factor 4, also known as mediumwasremovedandreplacedwithcompleteNeurobasal
CREB-2)andtheCCAAT/enhancer-bindingprotein(C/EBP(cid:1)) medium or with Krebs-Ringer bicarbonate buffer: 119 mM
hasbeenidentifiedintheSLC38A2genecodingforSNAT2(32, NaCl,5.9mMKCl,1.2mMMgSO ,2.5mMCaCl ,5.6mMglu-
4 2
33), and a role for SNAT2 as a nutrient amino acid sensor cose, 25 mM NaHCO , pH 7.4, for various periods of time at
3
(exertingitseffectupstreamofthesignalingmechanismsreg- 37°C.SomeexperimentswerealsoperformedusingEarle’sbal-
ulating the adaptive regulatory response) has been proposed anced salt solution: 117.4 mM NaCl, 5.3 mM KCl, 0.81 mM
(34,35).ThestimulationofSystemAby(cid:2)AAtreatmentmay MgSO ,1.8mMCaCl ,5.55mMglucose,26.2mMNaHCO ,pH
4 2 3
alternativelybestress-inducedandrelatedtocellvolumeres- 7.4.Variousconcentrationsofunlabeledaminoacidsordrugs
torationthroughanexpansionoftheintracellularaminoacid were also added at this time. For refeeding experiments, the
pool(36,37)(butseeRefs.38and39).Adaptiveregulationof Krebs-Ringerbicarbonatebufferwaschangedwithfreshbuffer
System A has not previously been demonstrated in neurons, containingthedesiredconcentrationofaminoacid.
which express both System A isoforms, SNAT1 and SNAT2. Immunocytochemistry—Primary neuronal cultures were
TheeffectofincubationwithSystemAsubstrates,particularly fixedoniceincold((cid:2)20°C)methanolfor10min,rinsedthree
glutamine, on neuronal levels of glutamine transporters timesinPBS,blockedin0.2%Triton,6%normalgoatserumin
SNAT1andSNAT2hasalsonotbeenexamined. PBS at room temperature for 1 h, and incubated for 3 h in
blockingbufferwitharabbitpolyclonalantibody(1:1000)made
EXPERIMENTALPROCEDURES
againstaglutathioneS-transferasefusionoftheNterminusof
PrimaryNeuronalCulture—Primaryneuronalcultureswere SNAT1residues1–63(1)orSNAT2residues1–65(2).Cells
preparedasdescribed(40,41)withminormodifications.Cer- werethenwashedinPBS,incubatedfor1hinsecondaryanti-
ebral cortices were removed rapidly from rat embryos at bodiescoupledtoAlexa-488(highlycross-adsorbedanti-rab-
embryonicday18understereomicroscopicobservation,inice- bit), diluted 1:200 in blocking buffer, washed, mounted with
cold 1(cid:3) PBS containing 6% glucose, and the meninges were ProlongGoldantifadereagent(Invitrogen),andviewed.Epif-
discarded. Cortical tissue was placed in Neurobasal medium luorescencemicroscopywasperformed,andimageswerecap-
containing 0.02% bovine serum albumin, 0.1% papain, and 5 turedusinganRTsliderSpotcameraunderidenticalexposure
mML-cysteineandincubatedwithgentleshakingat37°Cfor20 conditionsforcomparativeanalysis.
min.Thetissuepieceswerethenremoved,rinsedthreetimes WesternAnalysis—Westernanalysiswasperformedonpost-
withDMEMcontaining10%fetalbovineserumand2.5(cid:4)g/ml nuclearsupernatantfractionscontainingproteininhibitors(5
leupeptin, and dissociated by trituration using fire-polished (cid:4)g/mlpepstatin,5(cid:4)g/mlaprotinin,5(cid:4)g/mlleupeptin,1mM
Pasteur pipettes in complete Neurobasal medium supple- phenylmethylsulfonyl fluoride) following brief sonication and
mented with B27 (Invitrogen) and 0.5 mM Glutamax. Debris centrifugationat2,000(cid:3)g.Westernblottingwasperformedas
was allowed to settle for 10 min, and the cells in suspension described (43) using antibodies against SNAT1 (1:1000) and
were centrifuged for 10 min at 800 (cid:3) g and resuspended in SNAT2 (1:1000) and visualized using peroxidase-conjugated
completeNeurobasalmediumsupplementedwith25(cid:4)ML-glu- anti-rabbitIgGsecondaryantibodies(Sigma)andSuperSignal
tamate.Cellswerecountedinthepresenceoftrypanblueand West Pico chemiluminescent substrate (Pierce) followed by
platedin6-wellclusters(0.75(cid:3)106cells/well)precoatedwith exposuretofilm.
poly-D-lysine(25(cid:4)g/ml;Sigma).Cellsweremaintainedincul- Transport Assays in Cultured Neuronal Cells—We assayed
turebyrefreshingthemedium(50%)twiceaweekwithNeuro- SystemAfunctionalactivity(attributabletobothSNAT1and
basalmediumsupplementedwithB27and0.5mMGlutamax. SNAT2)attheplasmamembranebymeasuringuptakeof(cid:3)-[1-
TheaminoacidcompositionofNeurobasalmediumisasfol- 14C]methylaminoisobutyric acid ([14C]MeAIB; PerkinElmer
lows20(cid:4)Malanine,5(cid:4)Masparagine,10(cid:4)Mcysteine,500(cid:4)M LifeSciences)inprimaryneocorticalcellcultures.Weassigned
glutamine,400(cid:4)Mglycine,200(cid:4)Mhistidine,200(cid:4)Mmethio- toSystemAactivitythe50(cid:4)M[14C]MeAIBtransportactivity
nine, 67 (cid:4)M proline, 400 (cid:4)M serine, 800 (cid:4)M threonine. The sensitivetoinhibitionby4mMalanine,MeAIB,orglutamine.
overallconcentrationofSNAT2substratesinneuronalculture Toinitiateuptake,cellswerefirstrinsedwithuptakemedium
medium is 2.6 mM. Other chemicals not present in Krebs- (2 (cid:3) 1 ml) comprising 125 mM NaCl, 4.8 mM KCl, 1.2 mM
Ringer amino acid deprivation medium are as follows: 8 (cid:4)M KH PO ,1.2mMMgSO ,1.2mMCaCl ,5.6mMglucose,25mM
2 4 4 2
calciumpantothenate,28(cid:4)Mcholinechloride,8(cid:4)Mfolicacid, HEPES (pH 7.4) and then incubated at 37°C with uptake
40(cid:4)Minositol,30(cid:4)Mniacinamide,20(cid:4)Mpyridoxal,1(cid:4)Mribo- mediumcontaining1(cid:4)l/ml[14C]MeAIB(50.5mCi/mmol)and
flavin, 10 (cid:4)M thiamine, 0.2 (cid:4)M vitamin B12, 230 (cid:4)M sodium 50(cid:4)MunlabeledMeAIB(intheabsenceorpresenceofinhibitor
pyruvate,25,000(cid:4)MHEPES.Thetotalosmolalityofcomplete amino acids or drugs). At the end of the uptake period, we
APRIL24,2009•VOLUME284•NUMBER17 JOURNALOFBIOLOGICALCHEMISTRY 11225
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
removed the uptake medium, rinsed the cells with ice-cold end of each experiment to verify that a single product per
uptakemedium(2.5ml),andsolubilizedthecellsin1mlof1% primerpairwasamplified.ThesizeoftheamplifiedDNAfrag-
SDS.Fromthesesamples,weremoved30-(cid:4)laliquotsforpro- ment was verified by gel electrophoresis on a 3% agarose gel.
tein determination using the Micro BCA kit (Pierce); to the Samples were compared using the relative C method. The
T
remainder,weadded7.5mlofEcoScintandmeasured14Ccon- -foldincreaseordecreasewasdeterminedrelativetoavehicle-
tentbyliquidscintillationcounting. treatedcontrolafternormalizingtoahousekeepinggeneusing
ElectrophysiologicalRecordingsinCulturedNeuronalCells— 2(cid:2)(cid:7)(cid:7)CT,inwhich(cid:7)C represents(geneofinterestC )(cid:2)((cid:1)-ac-
T T
WholecellpatchclamprecordingsweremadeusingAxopatch tinC )and(cid:7)(cid:7)C is((cid:7)C treated)(cid:2)((cid:7)C control).The(cid:1)-ac-
T T T T
200Bamplifiers.Externalsolutionscontained140mMNaCl,5 tin-normalizing control was always included in the real time
mMKCl,1.25mMNaH PO ,10mMHEPES,2.5mMCaCl ,1.0 RT-PCRexperiments.
2 4 2
mMMgCl ,10mMglucose.Wholecellrecordingpipettes(3–5 ExpressionandAnalysisofSNAT2inXenopusOocytes—We
2
(cid:4)M) were pulled from borosilicate glass. The internal pipette performedlaparotomyandovariectomyonadultfemaleXeno-
solutionscontained120mMpotassiumgluconate,20mMKCl,4 pus laevis frogs under 2-aminoethylbenzoate methanesulfon-
mMNaCl,10mMHEPES,0.1mMEGTA,4.0mMMg ATP,0.3 ate anesthesia (0.1% in 1:1 water/ice, by immersion). Ovarian
2
mM Tris GTP, and 14 mM phosphocreatine (pH 7.25 with tissue was isolated and treated with collagenase A (Roche
2
KOH).Werecordedspontaneousfiringundercurrentclamp. Applied Science), and defolliculate oocytes were isolated and
Thefrequencyandamplitudeofminiatureexcitatorypostsyn- storedat17°CinmodifiedBarthsmedium,asdescribed(44).
aptic currents (mEPSC) recorded under voltage clamp (at a SNAT2wasexpressedinoocytesasdescribed(2).Weanalyzed
holding potential of (cid:2)70 mV) were obtained using the Mini- SNAT2 activity in oocytes by measuring amino acid-evoked
Analysissoftware.Bicuculline(10(cid:4)M)wasaddedtotheexter- currents and transport of 100 (cid:4)M [14C]MeAIB as described
nalsolutiontoeliminateGABAreceptor-gatedcurrentswhen (2,6).
measuring spontaneous excitatory activity. When mEPSCs Chemicals—Cycloheximide(CHX)wasdissolvedinethanol,
wererecorded,bothbicucullineandtetrodotoxin(1(cid:4)M)were and actinomycin D (ACD) was dissolved in DMSO. Solvents
includedintheexternalsolution. (0.05%)alonehadnoeffectonSNAT2expressionincontrolor
RealTimeRT-PCR—TotalRNAwasisolatedfrom0.75(cid:3)106 (cid:2)AA conditions (data not shown). Amino acids were pur-
cells using commercial silica-based filter binding methods chasedfromSigmaanddissolvedinwaterorbuffer,adjusting
(RNeasyminikit;Qiagen).Wereversetranscribed1(cid:4)gofRNA pH with NaOH when necessary. Radiochemicals were pur-
using a mixture of oligo(dT) and random hexamers (iScript chased from PerkinElmer Life Sciences. (S)-SNAP 5114 and
cDNA synthesis kit; Bio-Rad). Real time PCR was performed NNC711wereobtainedfromTocrisBioscience.
using the iCycler iQ detection system (Bio-Rad) according to StatisticalAnalyses—Dataareexpressedasmean(cid:8)S.E.from
themanufacturer’sinstructions.Reactionswereperformedin samplesofsizen.Statisticalanalyses(Student’sttest,nonlinear
triplicateina25-(cid:4)ltotalvolumewith2(cid:4)loftemplate,200nM regressionorone-wayanalysisofvariancefollowedbyposthoc
primers,and12.5(cid:4)lofSYBRgreenPCRMasterMix(Applied multiplecomparisonsusingtheHolm-Sidakmethod)wereper-
Biosystems).ThePCRcyclewasasfollows:95°Cfor8minand formedusingPrism3(Graphpad)orSigmaStat3.5(SystatSoft-
40cyclesof95°Cfor15sand60°Cfor45s.Theefficiencyofthe ware)withcriticalsignificancelevelp(cid:9)0.05.Nonlinearregres-
reactionforeachtargetwasfirsttestedusing10-foldserialdilu- sion analysis used the least squares method, and errors
tionsofplasmidcontainingthecognategene(104to109copies) presentedaretheS.E.oftheestimates.
to ensure (cid:5)90% efficiency. Specific primer sets, designed to
amplify(cid:4)200-bpfragments,wereforSNAT1(5-cgttgtttgagct- RESULTS
ggccaagaagacg (forward) and 5(cid:6)-gtattcttgtctccatcctggttcgtg SystemAUndergoesAdaptiveRegulationinPrimaryNeuro-
(reverse)), SNAT2 (5(cid:6)-gcatgaagaagaccgaaatgggaagg (forward) nal Cultures—To assess System A transport activity at the
and 5(cid:6)-cctggatgaaagtctgtttcgtacttc (reverse)), TauT (SLC6A6) plasmamembrane,weassayed[14C]MeAIBuptakeinratneo-
(5(cid:6)-tggcggtgataacttatatgacggtattg(forward)and5(cid:6)-gcagaggag- corticalneuron-enrichedculturesat12daysinvitro.Transport
gatgacaatgaccaag (reverse)), ATF4 (5(cid:6)-cctgactctgctgcttatat- of50(cid:4)M[14C]MeAIBwasinhibitedby(cid:4)95%inthepresenceof
tactctaac(forward)and5(cid:6)-actccaggtgggtcataaggtttg(reverse)), saturating (10 mM) cold alanine. The remaining fraction was
C/EBP(cid:1) (5(cid:6)-caacctggagacgcagcacaag (forward) and 5(cid:6)-ctag- largelyattributabletobackgroundlabeling,sowetooktheala-
cagtgacccgccgagg (reverse)), and (cid:1)-actin (5(cid:6)-taggcaccaggg- nine-sensitive [14C]MeAIB uptake as an index of System A
tgtgatggtggg (forward) and 5(cid:6)-cgcagctgattgtagaaggtgtggtg activity.WefoundthatSystemAtransportactivitywassignif-
(reverse)). 18 S primers (TaqMan Ribosomal RNA control icantly up-regulated 5-fold following 8 h of incubation in
reagents)werepurchasedfromPerkinElmerLifeSciences.The Krebs-Ringerbuffercontaining5.6mMglucose((cid:2)AA)(Fig.1).
PCRamplificationofeachproductwasfurtherassessedusing The increased System A transport activity was completely
10-folddilutionsofaratbraincDNAlibraryasatemplateand blocked in the presence of glutamine, alanine, or MeAIB (4
foundtobelinearover5ordersofmagnitudeand(cid:5)95%effi- mM).Weperformedkineticanalysesoftheuptakeprocessand
cient.Duplicatereactionsweresetupina25-(cid:4)ltotalvolume found that the increase in System A activity resulted from a
with5pmolofeachprimer,12.5(cid:4)lof2(cid:3)SYBRgreenMaster 2-fold increase in V following acute (1-h) (cid:2)AA treatment
max
Mix(AppliedBiosystems),and2(cid:4)loftemplate.ThePCRcycle (datanotshown)anda5-foldincreaseinV followinglong
max
was95°Cfor3min,42cyclesof95°Cfor30s,60°Cfor30s,and term(8-h)(cid:2)AAtreatment,withouteffectonK (Fig.1,Band
m
72°Cfor30s,andameltcurveanalysiswasperformedatthe C),consistentwiththeeffectsof(cid:2)AAtreatmentinperipheral
11226 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER17•APRIL24,2009
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
FIGURE 2. Induction of SNAT2 but not SNAT1 protein synthesis in
response to (cid:5)AA treatment. A, Western analysis of SNAT1 and SNAT2
expressionincontrol(Con)conditions(Neurobasalmedium),(cid:2)AA,and(cid:2)AA
inthepresenceofGln(4mM),CHX(75(cid:4)g/ml),orACD(7.5(cid:4)g/ml).Control
conditionswithACDarealsoshown.Blotswereprobedwithanti-SNAT1or
-SNAT2rabbitantibodies.B,selectiveup-regulationofSNAT2by(cid:2)AAtreat-
mentwasblockedbyglutamine.NotethatSNAT2inductionoccurredprinci-
pallyinneuronalsomataanddendriticprocesses.Resultsshownarefroma
representativeexperimentthatwasrepeatedinanindependentculturewith
similarresults.
The increase in SNAT2 protein was blocked by both agents
(Fig.2A),suggestingthat(cid:2)AAtreatmentinducesdenovosyn-
thesis of SNAT2 protein. These observations are consistent
FIGURE1.ThenumberoffunctionalSystemAtransportersontheplasma
membrane(V )isup-regulatedby(cid:5)AAtreatmentinneuron-enriched with the “derepression” of System A as previously described
max
cultures.A,initialvelocity(3min)ofMeAIBuptakeasafunctionofconcen- (12).AcutechangesinSystemAactivity(2-foldincrease)fol-
tration(0.1–3.0mM)incontrolandfollowinglongterm(8-h)(cid:2)AAtreatment. lowing(cid:2)AAtreatmentwerenotaffectedbyCHXorACD(data
BandC,datainAwerefittedbytheMichaelis-Mentenequationusingnon-
linearregression,revealinganincreaseinV following8-h(cid:2)AAtreatment not shown). We also visualized subtype-specific regulation of
(a,p(cid:9)0.001)withouteffectonKm(b,p(cid:1)0m.8ax3).Resultsshownaretheaver- SystemAbyfluorescencemicroscopywithconstantexposure
ages of duplicate measurements in one representative experiment; we
obtainedsimilarresultswhentheexperimentwasrepeatedinasecondneu- times (Fig. 2B). SNAT2 immunoreactivity was prominently
ronalpreparation. observedinmostneuronsinresponsetolongterm(cid:2)AAtreat-
mentbutnotinsimilarculturestowhichweadded4mMglu-
cells(12).SystemAactivityisthereforesubjecttoadaptivereg- tamine and not in control neurons (amino acid-replete). We
ulationinprimaryneuronalcultures. observednochangeinfluorescenceintensityforSNAT1incor-
Whereas a multiplicity of glutamine transport systems are tical neurons following long term (cid:2)AA treatment. Adaptive
expressedinratbrainprimarycultures,namelyNa(cid:10)-independ- regulation of System A in neurons is therefore selective for
entSystemL(45),Na(cid:10)-dependentSBAT1/B0AT2(SLC6A15), SNAT2.
(46,47),D-aspartatesensitivetransporter(48),andsystemA(7, Down-regulation of SNAT2 in the Presence of SNAT2
49),onlythatfractionofthe[3H]glutamineuptakethatisinhib- Substrates—Sincetheup-regulationofSystemAfollowinglong
ited by MeAIB was increased by (cid:2)AA treatment (data not term(cid:2)AAtreatmentisdueentirelytoSNAT2,weusedmanip-
shown).Therefore,SystemAistheonlyneuronalglutamine- ulationofincubationconditionstostudytheaminoacidspec-
transportsystemthatundergoesadaptiveregulationfollowing ificityofSNAT2adaptiveregulationinprimaryneurons.Glu-
aminoaciddeprivation. tamineat4mMcompletelyreversedthelongterm(8-h(cid:2)AA
Adaptive Regulation of System A in Neurons Is Isoform- treatment)adaptiveregulationofSNAT2(Fig.2),andat(cid:4)0.5
specific—Centralnervoussystemneuronsexpresstwoisoforms mM,glutamineresultedin50%repression(seeTable1andFig.
ofSystemAtransporter,soweexaminedthelevelofexpression 7).TheIC valuesestimatedforrepressionofSNAT2expres-
50
of SNAT1 and SNAT2 protein following (cid:2)AA treatment. By sion by glutamine (Table 1) and also by alanine (data not
Western analysis, SNAT1 immunoreactivity levels were rela- shown) matched the half-maximal concentrations we have
tivelyconstant,whereasSNAT2proteinlevelswereselectively observedforSNAT2-andSNAT1-mediatedtransportofthese
up-regulated in response to 8 h of(cid:2)AA treatment (Fig. 2A). substrates(i.e.K of0.2–0.5mM)(1–3,50).
0.5
Thisup-regulationofSNAT2wascompletelyabolishedinthe SNAT2ExpressionLevelsDoNotAffectSpontaneousGluta-
presence of 4 mM glutamine. To determine whether the matergicActivity—Havingdeterminedthatwecanmanipulate
increaseinSystemAactivityfollowinglongterm(cid:2)AAtreat- SNAT2expressionlevelsbyexploitingtheadaptiveregulation
mentwasduetochangesinmRNAandproteinsynthesis,we propertyofSNAT2,wetestedwhetherSNAT2playsanimpor-
examinedtheeffectsofCHX(75(cid:4)g/ml)orACD(7.5(cid:4)g/ml). tantroleinprovidingexcitatoryneuronprecursorsforneuro-
APRIL24,2009•VOLUME284•NUMBER17 JOURNALOFBIOLOGICALCHEMISTRY 11227
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
FIGURE3.AlteringendogenousSNAT2levelsdoesnotaffectspontaneousexcitatoryactionpotentialsinneuronalculturedcells.A–E,typicaltraces
recordedfromneuronspretreatedfor8hwithcontrol(cid:10)0.5mMGln,control(cid:10)4mMGln,(cid:2)AA,(cid:2)AA(cid:10)4mMGln,and(cid:2)AA(cid:10)4mMMeAIB.Spontaneousaction
potentialswereeliminatedinthepresenceofDNQX(10(cid:4)M),anAMPAglutamatereceptorantagonist.F,meanfrequenciesofspontaneousfiring(normalized
tothecontrol)undervariousconditions(a,p(cid:9)0.05versus(cid:2)AAtreatment).Con,control.
transmitterglutamatebymeasuringspontaneousglutamater- control and (cid:2)AA conditions (Fig. 3, D–F). Since System A
gicactivityinneuronalculturesinthepresenceandabsenceof transport is rheogenic (2, 6, 52), currents can be detected by
aminoacids.Althoughlowinabundance(andnondividingin transientapplicationofglutaminetoneurons(5).Theeffectof
Neurobasalmedium),astrocyteswerepresentinourcultures MeAIBwaslessrobust(Fig.3,CandF),andthismayrelateto
(GFAPstaining)toprovideendogenousglutaminetoneurons thefactthat,whereasMeAIBisaspecificsubstrateofSystemA,
forneurotransmission(datanotshown).Weusedratneocorti- it is poorly transported, particularly true for SNAT1 (6). We
calneuron-enrichedcultures(12daysinvitro)duringthemajor foundnorelationshipbetweenSNAT2levelsandspontaneous
developmentalperiod(7–18daysinvitro)inwhichfunctional action potentials. Glutamine also enhanced the frequency of
maturation of glutamate vesicular storage and release in syn- mEPSCs(Fig.3,A–C)andspontaneousactionpotentialfiring
apsesoccurs(42)inordertoexaminetherelationshipbetween (Fig.4,DandE);however,wefoundnodifferenceintheampli-
SNAT2expressionandspontaneousglutamatergicactivity.We tudeofmEPSCsfollowing(cid:2)AAtreatmentorinthepresenceof
firstexaminedthespontaneousglutamatergicactivityofneu- 4mMglutamine(Fig.4,BandC).
rons in control cultures and following adaptive regulation to SNAT1 mRNA Is More Stable than Is SNAT2 mRNA—We
assessactiveneuronalconnectivityandintegrityofourprep- assessed the molecular response of adaptive regulation of
arations and to correlate levels of SNAT2 and glutamine SNAT2inneuronsbyrealtimeRT-PCR.SNAT2mRNAlevels
withexcitatorytransmission.Wefoundnodifferenceinthe increased(cid:4)10-foldfollowing(cid:2)AAtreatment,whereasSNAT1
frequencyofspontaneousactionpotentialsorthefrequency mRNA expression was unchanged (Fig. 5A). SNAT1 mRNA
andmagnitudeofmEPSCsbetweencontrolneuronsandneu- wasmorestablethanSNAT2mRNAundercontrolconditions
ronsdeprivedofaminoacids(includingglutamine)for8h(Fig. andwasnotaffectedbyadaptiveregulation(Fig.5B).SNAT2
3,A,B,andF).Theseresultsareconsistentwitharecentreport mRNA stability is increased significantly in (cid:2)AA conditions
indicatingthatglutaminesuppliedexogenouslyisnotnecessary (Fig.5C).NotingalsothatSNAT2proteinstabilityisincreased
for glutamatergic transmission in acute hippocampal slices by (cid:2)AA treatment in muscle cells (35), we speculate that
(51). In contrast, incubation with 4 mM glutamine for 8 h SNAT2proteinlevelsshouldbelowintheabsenceofdenovo
resulted in a marked increase in spontaneous firing in both SNAT2mRNAsynthesis.
11228 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER17•APRIL24,2009
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
FIGURE4.GlutamineenhancesmEPSCs.A,representativesweepsofmEPSCsrecordedinculturedcorticalneuronstreatedwithvehicle,Gln(4mM),(cid:2)AA,and
(cid:2)AA(cid:10)Gln(4mM).Themembranepotentialwasheldat(cid:2)70mV.Bicuculline(10(cid:4)M)andtetrodotoxin(TTX)(0.5–1(cid:4)M)wereincludedintheexternalsolution.
ThesynapticeventswereanalyzedusingtheMiniAnalysisprogram.B,cumulativeprobabilityofmEPSCamplituderecordedinneuronsunderdifferent
treatments.C,meanpercentagechangesintheamplitudeofmEPSCs.D,cumulativeprobabilityofmEPSCfrequency.E,meanpercentagechangesinthe
frequencyofmEPSCs.a,p(cid:9)0.05comparedwiththevehiclecontrol;b,p(cid:9)0.05comparedwith(cid:2)AAtreatment.
Rapid Down-regulation of SNAT2 in Response to Refeeding
System A Substrates—We assessed SNAT2 stability following
adaptiveregulationinducedbyglutaminerefeeding(4mM).We
used as a reference the non-System A substrate taurine, an
importantosmolyteinneurons.Weobservedmorerapiddis-
appearanceofSNAT2proteininresponsetoglutaminethanto
taurine (Fig. 6). At 2 h ofincubation, glutamine repressed
SNAT2mRNAexpressionmorestronglythantaurine,butat
5h,bothaminoacidsreducedSNAT2mRNAtocontrollevels
(Fig. 6A). Taurine also down-regulated SNAT2 protein and
activity; however, the effect was delayed compared with the
effectofglutamine(Fig.6,BandC).Ourresultsthereforeindi-
catethatSNAT2proteinhasarelativelyshorthalf-lifeinthe
presenceofSystemAsubstrates.
Reexamination of the Substrate Profile of System A—Since
8-h refeeding with taurine resulted in the disappearance of
FIGURE5.SNAT2mRNAstabilityisconstitutivelylowbutincreasedfol-
SNAT2 mRNA and protein expression (Fig. 6, A and B) and lowing(cid:5)AAtreatment.A,realtimeRT-PCRshowsthatSNAT2mRNAwas
System A functional activity (Fig. 6C), we reexamined the selectivelyanddramaticallyup-regulatedcomparedwithSNAT1mRNAfol-
lowing(cid:2)AAtreatment.B,SNAT1mRNAstabilitywasrelativelyconstantfol-
gamutofaminoacidscapableofadaptivelyregulatingSystemA lowingtheadditionofACDandunaffectedby(cid:2)AAtreatment.C,SNAT2
inneurons.Inadditiontotaurine,wetestedthesimilarcom- mRNAstabilitywaslowincontrolconditionsandgreatlyincreasedin(cid:2)AA
pounds (cid:1)-alanine and (cid:1)-aminoisobutyric acid ((cid:1)-AIB), other treatment.Startinglevelsareatt(cid:1)0aftertheadditionofACD.ResultsinAare
fromthreeindependentcultures.ResultsinBandCareaveragesofduplicates
known osmolytes (betaine, myo-inositol, and sorbitol), and intwoindependentexperiments.Con,control.
APRIL24,2009•VOLUME284•NUMBER17 JOURNALOFBIOLOGICALCHEMISTRY 11229
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
stratesglutamineandalanineinblockingtheadaptiveincrease
inSNAT2mRNA(Fig.7,AandB).myo-Inositolandsorbitoldo
notrepressSNAT2expression,indicatingthatonly(cid:3)-and(cid:1)-
aminoacidsareeffective.
These findings prompted us to examine whether SNAT2
maybecapableofalsotransportingtaurine,GABA,or(cid:1)-ala-
nine, so we extended our previous analyses (2, 6) of the sub-
strateprofileofSNAT2tothesecandidatesubstrates.Over-
expression of SNAT2 in Xenopus oocytes stimulated the
uptakeof100(cid:4)M[14C]MeAIB60-foldoverthatobservedin
control oocytes (Fig. 8A). SNAT2-mediated [14C]MeAIB
transportwasinhibited81(cid:8)1%by1mML-alanine,whereasno
inhibitionwasobservedinthepresenceof(cid:1)-alanine,GABA,or
taurine(eachat1mM).WhereasL-glutamineandL-alanine(10
mM)evokedlargecurrentsinoocytesexpressingSNAT2(Fig.
8B),taurineevokednocurrentandGABA,onlyatinycurrentif
atall.InoocytesexpressingSNAT2,(cid:1)-alanineevokedonlyvery
small currents that were (in three oocytes) 7.5 (cid:8) 1.6% of the
currentevokedbyL-alanine.Thesedatademonstratethattau-
rineandGABAarenotSNAT2substratesandsuggestthatno
significant transport of (cid:1)-alanine (at best a weak substrate of
SNAT2) will proceed in the presence of physiological gluta-
mineconcentrations.WealsofoundthatGABAand(cid:1)-alanine
werenotsubstratesforSNAT1(datanotshown).
TodeterminewhethertheregulationofsystemAbynonsub-
stratesisuniquetoneuronsorisobservedinotherperipheral
cells,weexaminedtheeffectof(cid:2)AAtreatmentinHeLacells,
a human epithelial carcinoma cell line. We found that
[14C]MeAIB uptake increased 5-fold following 8 h of(cid:2)AA
treatment,whichwasblockedby4mMglutamineorMeAIB,as
in neurons. However, taurine and GABA (4 mM) could not
repressadaptiveregulationofsystemAfollowing(cid:2)AAtreat-
mentinHeLacells(datanotshown).
Regulation of SNAT2 by Transporters in the SLC6 GABA
Transporter Subfamily—Transporters in the GABA trans-
portersubfamily(SLC6)includethoseservingforthetransport
ofneurotransmitters(GABA),osmolytes(taurine,betaine,and
GABA),andfuel(creatine)(53,54).WefoundthatGABA,(cid:1)-al-
anine,andtaurineinhibitedtheup-regulationofSNAT2with
roughlyequivalentIC values((cid:9)50(cid:4)M),whichwereone-tenth
50
theIC forglutamine((cid:4)500(cid:4)M)(Fig.7andTable1).Wehave
50
rankedinTable1thepotenciesofarangeofpotentialSystemA
repressorsatthemRNAlevel(fromdatainFig.7,AandB)and
FIGURE6.ThedecayinSNAT2mRNA,totalprotein,andfunctionalactiv- thefunctionallevel(fromdatainFig.7,CandD).
ityinresponsetoglutaminerefeedingof(cid:5)AA-treatedculturesisfaster
SinceGABAisamajorinhibitorytransmitterinthebrain,we
thanbythenon-SystemAsubstratetaurine.Cellswereaminoacid-de-
privedfor8h,andthenfresh(cid:2)AAmediumcontaining4mMglutamineor investigated whether its ability to repress SNAT2 expression
taurinewasadded.CellsweresampledformRNA,protein,andfunctional may be GABA receptor-mediated. We found that the 90%
analyseswithtime.A,realtimeRT-PCRanalysisofSNAT1andSNAT2mRNA
reduction in SNAT2 expression (at the level of RNA and
levelsfollowingglutamineortaurinerefeedingofculturespre-exposedto
(cid:2)AAconditionsfor8h.B,Westernblotanalysis;C,[14C]MeAIBuptakefollow- [14C]MeAIBtransportactivity)inducedby200(cid:4)MGABAwas
ing8-h(cid:2)AAtreatmentandrefeedingwithglutamineortaurine.Resultspre- unchanged in the presence of the GABA receptor blockers
sentedinAandCareaveragesoftwoindependentexperiments. A
bicuculline (100 (cid:4)M) or picrotoxin (100 (cid:4)M) or the GABA -
B
receptor blocker baclofen (100 (cid:4)M) (data not shown). There
GABA. When neuronal cultures were incubated with these existintheratfourdistinctproteinsencodingGABAtransport-
compounds(at1mMinKrebs-Ringerbicarbonatebuffer)(Fig. ers, namely GAT-1 (GABA transporter 1; SLC6A1), GAT-2
7,AandC),wefoundthateachoftheseaminoacids(allsub- (GABAtransporter2;SLC6A13),GAT-3(GABAtransporter3;
strates of the SLC6 GABA transporter subfamily) effectively SLC6A11), and BGT-1 (betaine-sensitive GABA transporter
repressSNAT2(Fig.7).Infact,GABA,(cid:1)-alanine,(cid:1)-AIB,and (SLC6A12)) (53, 54). In our neuronal cultures, GABA trans-
taurinearemorepotentthanarethepreferredSystemAsub- porter-selective compounds block only a portion of the total
11230 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER17•APRIL24,2009
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
(cid:4)M).Instead,theGAT-2/3-specific
blocker,SNAP-5114(100(cid:4)M)sup-
presses by (cid:4)50% the repression of
SNAT2mRNAby(cid:1)-alanineand,to
a lesser extent, that induced by
GABA (data not shown). Whereas
(cid:1)-alanine is not a substrate for
GAT-1,itistransportedbyGAT-2
andGAT-3andweaklytransported
by BGT-1 (57–59). Hence, GABA
and (cid:1)-alanine are probably being
taken up through the same GABA
transporters, which may include
GAT-3,andsuppressadaptivereg-
ulation of SNAT2. The taurine
transporter also efficiently trans-
ports(cid:1)-alanine(60).Sincetaurineis
the most potent repressor of
SNAT2mRNA(Fig.5andTable1),
we conjecture that the action of
(cid:1)-alaninetorepressSNAT2induc-
tion can be accounted for both by
GABAtransportandtaurinetrans-
portsystemsinneurons.Betaine,a
substrate of BGT-1 and a well rec-
ognizedosmolyteinthekidney(61),
also represses SNAT2 mRNA
induction, but higher concentra-
FIGURE7.RepressionofSNAT2byneutralaminoacidosmolytesoftheSLC6GABAtransportersubfam-
ily.A,adaptiveregulationwasnotrestrictedtoSystemAsubstrates.TotalcDNAwaspreparedfrommRNA tionsarerequired(Fig.7).Infurther
isolatedfrom(cid:2)AA-treatedculturesincubatedwithvariousaminoacids,aminoacidanalogues,andother supportofNa(cid:10)-coupledandsolute
compoundsfor8h.RealtimeRT-PCRofcDNA(1(cid:4)g)wasusedtoassesstheactivityandrelativepotencyof
thesecompoundstorepressSNAT2mRNAinduction.B,dose-responsecurvesofneutralaminoosmolytesof co-transport-mediated regulation
theSLC6GABAtransportersubfamily,comparedwithglutamine,addedtoculturesfollowing(cid:2)AAtreatment. ofSNAT2expression,wefoundthat
C,thereductionofSNAT2functionalactivitybyincubatingcultureswiththesevariouscompoundsfollowed
transporter-selective substrates (1
thesameorderasSNAT2mRNArepressioninA.D,dose-responsecurvesoftheeffectofneutralaminoacid
osmolytesofSLC6GABAtransportersubfamilyandglutamineonreducingSNAT2functionalactivityin(cid:2)AA mM) such as nipecotic acid (a sub-
conditions. strate for GAT-1 and GAT-3
(mouse GAT-4) (58)) and guanidi-
TABLE1 noethylsulfonate (a substrate of the taurine transporter (62))
PotencyofvariouscompoundstorepressSNAT2 areeffectiverepressorsofSNAT2up-regulationatthemRNA
IC a level(Fig.7A)and,lessso,attheproteinlevel(Fig.7C).
Compound 50
RT-PCR (cid:11)14C(cid:12)MeAIB RegulationofSNAT2expressioninneuronsthereforecom-
(cid:4) prisesrepressionofSNAT2mRNAsynthesisbynonsubstrates,
M
Taurine 19 18 namelyGABA,andseveral(cid:1)-aminoacids,suchastaurine,(cid:1)-al-
(cid:1)-Alanine 28 14 anine,and(cid:1)-AIB,andaccelerationofSNAT2mRNAandpro-
GABA 39 16
Glutamine 496 570 teindegradationbySystemAsubstrates.Thus,SNAT2expres-
Betaine 819 1497
sion is expected to be constitutively low under normal
aDataareaveragesoftwoindependentexperiments.
conditions but potently induced in response to depletion of
neutral(cid:3)-and(cid:1)-aminoacids.
[3H]GABAtransport;GAT-1(NNC-711-sensitive)andGAT- MechanismofRepressionofSNAT2mRNAInductionbyGlu-
2/3 (SNAP-5114 sensitive) each comprise at least 40%, and tamineandTaurineinNeurons—TodeterminewhetherNa(cid:10)-
BGT-1 (betaine-sensitive) only a minor portion (10%) of the coupled transport of solutes by neutral amino acid osmolyte
total[3H]GABAtransport(datanotshown).Interestingly,GAT-3 transportersoftheSLC6GABAsubfamilyleadstorepression
is abundantly expressed in pyramidal neurons during develop- of the SLC38A2 gene coding SNAT2 in neurons in the same
mentinratbrainandaccountsforalargefractionofGABAtrans- way as does System A-mediated transport of glutamine, we
portinvivo(55).GAT-1,theprincipleGABAreuptaketransporter examinedthemechanismofSNAT2mRNArepression.Previ-
in GABAergic nerve endings (56), does not appear to be ous studies have indicated that ATF4 mRNA transcription is
involvedinGABAblockadeofincreasedSystemAactivityseen increased by adaptive regulation. This transcription factor
in adaptive regulation, because the GAT-1-specific blocker binds to the AARE of SNAT2 and is selectively repressed by
NNC711(100(cid:4)M)couldnotreversetheeffectofGABA(200 SystemAsubstrates(32).WefoundthattheinductionofATF4
APRIL24,2009•VOLUME284•NUMBER17 JOURNALOFBIOLOGICALCHEMISTRY 11231
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
Asubstratesasrepressors.AmongtheGABAtransporters,only
BGT-1 (also known as mGAT-2) mRNA levels were signifi-
cantly up-regulated ((cid:4)2-fold) by (cid:2)AA treatment in our cul-
tures(datanotshown);however,TauTmRNAlevelsincreased
(cid:4)6-fold following prolonged (cid:2)AA treatment (Fig. 9B). As
expected,theinclusionoftaurineinKrebs-Ringerbicarbonate
bufferblockedtheinductionofTauTmRNA(Fig.9B).Surpris-
ingly,theinclusionofglutaminealsoblockedadaptiveregula-
tion of TauT (Fig. 9B). Induction of both SNAT2 and TauT
mRNA is blocked by actinomycin D (data not shown) and
cycloheximide (Fig. 9B). Similar results were also observed
using Earle’s balanced salt solution as the (cid:2)AA treatment
instead of Krebs-Ringer bicarbonate buffer (Fig. 9C). Collec-
tively, our results indicate that these neutral (cid:3)- and (cid:1)-
aminoacidsmayserveacommonroleincentralneuronsthat
convergeatthelevelofgeneregulationoftheirrespectiveneu-
ronaltransporters.
DISCUSSION
AdaptiveRegulationofSNAT2mRNAExpressionbyAmino
Acid Deprivation in Neurons Is Not Selective for System A
FIGURE8.Aminoacidtransportandevokedcurrentsinoocytesoverex- Substrates—We have demonstrated for the first time that
pressingSNAT2.A,uptakeof100(cid:4)M[14C]MeAIBwasmeasuredover20min
(in100mMNaClmedium,pH7.5)incontroloocytesandoocytesexpressing SNAT2issubjecttoadaptiveregulationfollowingtotalamino
SNAT2;amongSNAT2:a,p(cid:1)0.003;b,notsignificant(p(cid:5)0.23)compared aciddeprivationinneuronsandthatsuchadaptiveregulation
withnoinhibitoraminoacid.B,aminoacid-evokedcurrentsinanoocyte
ofSNAT2isnotlimitedtotheeffectsofSystemAsubstrates.In
expressingSNAT2(i)andacontroloocyte(ii).Oocytesweresuperfusedin100
mMcholinechloridemedium(pH7.5)andthen,fortheperiodsshownbythe fact,substratesoftheSLC6GABAtransporterfamily,including
hatchedbars,inNaClmedium.Aminoacids(10mM)weresuperfusedforthe GABA, (cid:1)-alanine, (cid:1)-AIB, and taurine, block SNAT2 mRNA
periodsshownbythesolidbars.(TauandGABAevokednocurrentsinthis
induction(cid:4)10-foldmorepotentlythandoSystemAsubstrates.
controloocyte(datanotshown).)
We confirmed, in the oocyte overexpression system, that
mRNA((cid:4)50%)wasrepressedbyglutamineortaurinefollowing GABAandthe(cid:1)-aminoacidsdescribedarenotSystemAsub-
(cid:2)AAinneurons(Fig.9A). strates.Notsurprisingly,weuncoveredanearlyreportindicat-
Toexaminewhethermultiplepathwaysmayconvergeupon ingthat(cid:1)-alaninerepressestheadaptiveregulationofSystemA
the SNAT2 promoter during the adaptive response to (cid:2)AA inChinesehamsterovarycells(65),andanevenearlierreport
treatment in neurons, we examined the mRNA expression of showedthatthehydrophobicaminoacidstryptophan,pheny-
other related transcription factors. The AARE sequence (5(cid:6)- lalanine,andtyrosine(butnotleucine)couldrepressadaptive
ATTGCATCA-3(cid:6)) shows significant homology with the spe- regulationofSystemAinanestablishedlineofrathepatoma
cific binding sequences of two transcription factor families, cells(66).Nevertheless,confusionastothenatureofcontrolof
namelyATF4/CREB-2andC/EBP(cid:1)(33).Wethereforeexam- SNAT2expressionisstillapparentintheliterature.(cid:1)-Alanine
inedtheexpressionofC/EBP(cid:1)following(cid:2)AAtreatmentand andGABAaresubstratesofthetaurinetransporterTauT(67,
itsrepressionbyglutamineandtaurine.LikeATF4,wefindan 68) as well as of rat GAT-2/3 (57–59); orphan transporters
increase ((cid:4)2-fold) of C/EBP(cid:1)mRNA following (cid:2)AA treat- whosesubstratesincludeGABAandtaurinemayalsoexist(69).
ment,andthisisrepressedbyglutamineandtaurine(Fig.9A). In support of transport-mediated regulation of SNAT2, we
InductionofbothATF4andC/EBP(cid:1)following(cid:2)AAtreatment foundthattransporter-selectivesubstrates,suchasguanidino-
isblockedbyactinomycinDasexpected(datanotshown)but ethylsulfonate (taurine transporter), nipecotic acid (GABA
notbycycloheximide(Fig.9A).Bothtranscriptionfactorsare transporter),andMeAIB(SystemA),butnot2-aminobicyclo-
increasedby(cid:2)AA,andthebZIPfamilyoftranscriptionfactors (2,2,1)-heptane-2-carboxylicacid(SystemL),repressedadapt-
maybindtogenomicelementsasheterodimers(63,64).Thus, iveregulationofSNAT2.
ATF4-C/EBP(cid:1)maybothberequiredforSNAT2geneinduc- Our present findings support the concept that amino acid
tioninneurons.Blockadeoftheincreasedexpressionofeither deprivation releases neuronal SNAT2 from its constitutive
transcription factor (or both) may be sufficient to repress repression(i.e.“derepression”)atthegenomiclevel.Wefound
SNAT2mRNAsynthesis. thatthisadaptiveregulationisapropertyonlyofSNAT2and
Adaptive Regulation of the Taurine Transporter TauT Is not of SNAT1. Amino acid deprivation results in parallel
RepressedbyTaurineandGlutamine—Sincetransportedsub- increasesinSNAT2mRNA,plasma-membraneproteinexpres-
stratesoftheaminoacidosmolytetransporterswithintheSLC6 sion,andSystemAactivity(V effect).Thepresenceofany
max
GABAtransportersubfamilyarepotentrepressorsofSNAT2, SystemAsubstrateatsaturatingconcentrationsintheextracel-
weexaminedwhetherGABAtransportersorthetaurinetrans- lular medium blocks this up-regulation of SNAT2. The mid-
porter(TauT)alsoexhibitadaptiveregulationfollowing(cid:2)AA pointofglutamine-inhibitedSNAT2up-regulationinneocor-
treatmentinneuronsandassessedtheeffectivenessofSystem ticalneuronsoccursat(cid:4)0.5mM,whichlieswithintherangeof
11232 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER17•APRIL24,2009
SNAT2,an(cid:2)-AminoAcidOsmolyteTransporterinNeurons?
K values reported for SNAT2 (2, 3, 50) and is close to the
0.5
concentrationofglutamine((cid:4)0.4mM)expectedinextrasynap-
ticspace(14–16).Glutamineisfarmoreabundanttherethanis
either taurine or GABA, but glutamine repressed SNAT2 far
lesspotently.Taurineispresentinextraneuronalspaceatcon-
centrationsof35–50(cid:4)M(70).Attheseconcentrations,taurine
inhibitstheinductionofSNAT2activityinoursystem,suggest-
ingthattaurinemaynegativelyregulateexpressionofendoge-
nous SNAT2 mRNA levels in the brain. We therefore expect
that extracellular levels of glutamine and taurine (and also
GABA during development) will determine neuronal SNAT2
expressionlevels.
NeurotransmitterGlutamateRegenerationDoesNotRelyon
SNAT2—Spontaneousactivityofexcitatoryprojections(which
comprisethemajorityofcellsinourculture)isunaffectedby
removalofexogenousaminoacidsfor8h.Similarresultswere
recently obtained using acute hippocampal slices, indicating
thatpyramidalneuronshavethecapacitytoproduceglutamate
for long periods of time (51). Whereas resident astrocytes in
culture(andslices)mayexportendogenousglutaminedirectly
topyramidalneuronsviaSystemN(SNAT3,SNAT5)(20,21,
71),inourpreparation,ourdataindicatethatSNAT2doesnot
provideglutaminetoneuronsforglutamateneurotransmitter
regenerationundertheseconditions,becauseinducingSNAT2
levels((cid:5)5-fold)orrepressingSNAT2levelstonegligiblelevels
hadnoeffectonspontaneousactionpotential-derivedexcita-
torypostsynapticpotentials.Furthermore,anyincreaseincyto-
plasmicglutamatelevelsderivedfromglutamineimportedby
SNAT2wouldhavebeenexpectedtoincreasethequantalsize
ofglutamaterelease(72,73),butminiaturequantalamplitude
wasnotalteredfollowingSNAT2inductionorrepression.
WehavepreviouslyshownthatSNAT1andSNAT2protein
expressioninneuronsisrestrictedtosomataanddendriticpro-
cesses,andlittle,ifany,co-localizationwithsynaptophysin,a
synapticvesiclemarker,isapparent(6,8,10).Althoughgluta-
minetakenupinneuronalsomataanddendritesmaybecon-
verted to glutamate, the glutamate generated there is not
readilyavailabletosynapticvesiclesinaxonterminalsofpro-
jectionneuronstowhichthemajorvesicularglutamatetrans-
porter proteins VGLUT1 and VGLUT2 are restricted (43).
Indeed,Broerandco-workers(74)haveshownbyNMRspec-
troscopy that System A-mediated glutamine transport sup-
ports a large pool of neuronal glutamate that turns over very
slowly (presumably in cell soma and proximal dendritic
regions),andinstead,aseparatehistidine-sensitiveglutamine
transport system may be involved in providing glutamine for
excitatorytransmission.However,thepresenceofaglutamine
transporterinnerveterminalsthatsupportsglutamatetrans-
missionhasnotbeenestablished.Ourdatadonotruleoutarole
but both were repressed in the presence of 4 mM glutamine or taurine.
B,SNAT2andTauTmRNAexpressionincreasedfollowing(cid:2)AA,butbothwere
repressedinthepresenceof4mMglutamineortaurine.Resultspresentedare
themean(cid:8)S.E.offiveindependentexperiments.a,p(cid:9)0.01comparedwith
thevehiclecontrol;b,notsignificant(p(cid:5)0.05)comparedwiththevehicle
control.C,bothglutamineandtaurinerepressedSNAT2andTauTinduction
following(cid:2)AAtreatmentusingEarle’sbalancedsaltsolution.Insets,ATF4
FIGURE9.RepressionoftheinductionoftranscriptionfactorsATF4and andC/EBP(cid:1)mRNAinductionwasrepressedbyglutamineandtaurinesimi-
C/EBP(cid:3)andthetransportersSNAT2andTauTbyglutamineandtaurine. larlyasACD(7.5(cid:4)g/ml).Dataarefromarepresentativeexperimentper-
A,ATF4andC/EBP(cid:1)mRNAexpressionincreasedfollowing(cid:2)AAtreatment, formedintriplicate.
APRIL24,2009•VOLUME284•NUMBER17 JOURNALOFBIOLOGICALCHEMISTRY 11233