Proc. Natl. Acad. Sci. USAVol. 91, pp. 5158-5162, May 1994Pharmacology

Cloning, expression, and localization of a chloride-facilitated,cocaine-sensitive serotonin transporter from Drosophila melanogasterLIDIA L. DEMCHYSHYN*t, ZDENEK B. PRISTUPAt*, KIM S. SUGAMORI*t, ERIC L. BARKER§,RANDY D. BLAKELY§, WILLIAM J. WOLFGANG¶, MICHAEL A. FORTES, AND HYMAN B. NIZNIKtHIDepartments of tPsychiatry and *Pharmacology, University of Toronto, Toronto, ON M5S 1A8, Canada; §Department of Anatomy and Cell Biology, EmoryUniversity School of Medicine, Atlanta, GA 30322; lVollum Institute for Advanced Biomedical Research, Portland, OR 97201; and tLaboratory ofMolecular Neurobiology, Clarke Institute of Psychiatry, Toronto, ON M5T 1R8, Canada

Communicated by Avram Goldstein, February 16, 1994

ABSTRACT We report here on the isolation and charac-terization of a serotonin (SHT) transporter from Drosophilamelanogaster. A 3.1-kb complementary DNA clone (dSERT)was found to encode a protein of 622 amino acid residues witha predicted molecular mass of w69 kDa and a putative trans-membrane topology characteristic of cloned members of themammalian Na+/Cl- neurotransmitter cotransporter genefamily. dSERT displays highest overall amino acid sequenceidentity with the mammalian 5HT (51%), norepinephrine(47%), and dopamine (47%) transporters and shares with alltransporters 104 absolutely conserved amino acid residues.Upon tnsient expression in HeLa cells, dSERT exhibitedsaturabl, high-affinity, and sodium-dependent [3H5HT up-take with esimated K and V. values of w500 nM and 5.2 x1o-i8 mol per cell per min, respectively. In marked contrast tothe human SERT (hSERT), SHT-mediated transport bydSERT was not absolutely dependent on extracellular Cl-,while the sodium-dependent uptake of MHT was facilitatd byIncreased extracellular Cl- concentrations. dSERT disys apharmacological profile and rank order of potency consistentwith, but not identical to, mammalian 5HT transporters.Comparison of the afnities of various compounds for theinhibiion of 5HT transport by both dSERT and hSERTrevealed that antidepressants were 3- to 300-fold less potent ondSERT than on hSERT, while mazindol displayed w30-foldgreater potency for dSERT. Both cocaine and RTI-55 inhibited5HT uptake by dSERT with estmated inhibition constants of500 nM, while high concentrations (>10 FM) of dopmin,

norepinephrine, octopamine, tramine, and histamine failed toinhibit transport. In situ hybridization reveals the selectiveexpression of dSERT mRNA to specific cell bodies in theventral gagulon of the embryonic and larval Drosophila ner-vous system with a distribution pattern virtually identical tothat of 5HT-containing neurons. The dSERT gene was mappedto position 60C on chromosome 2. The availability of the genee ing the unique ion dependence and pharmacologicalcharacteristics of dSERT may allow for identification of thoseamino acid residues and structural motifs that confer thephannacologrc specificity and genetic regulation of the 5HTtransport process.

Serotonin (5HT) plays a vital role in modulation ofa variety ofbiochemical and physiological functions in the central nervoussystem, including sleep, appetite, and pain perception, and hasbeen shown to regulate complex behavioral phenomena, suchas learning and memory in invertebrates (1, 2). Numerousstudies have provided evidence for production of SHT inDrosophila as well as its cell-specific localization in neuronswithin the central nervous system (3). Released from neuronalterminals into the synaptic cleft, 5HT is believed to mediate its

effects in Drosophila via an interaction with specific multiplecell surface 5HT receptors (4, 5), which in turn are coupled tosubtype-specific guanine nucleotide-binding proteins (see ref.6 and references therein) activating various membrane effectorsystems. Removal of5HT from the synaptic cleft must also becarefully regulated for effective cellular responses, and severalstudies have documented the existence of various uptakesystems, including 5HT transporters (SERTs), in inverte-brates and Drosophila (7-10).

In mammals, the removal of neurotransmitters from thesynaptic cleft is mediated by members of a gene family ofNa+-dependent transporters displaying similar structural to-pology and ionic requirements. These include genes encodingtransporters for the biogenic amines SHT, dopamine (DA),and norepinephrine (NE) (11-18). The study ofthe molecularstructure and function of these transporters is clinicallyrelevant since substances of abuse, such as cocaine andamphetamines, as well as antidepressants, are thought toexert their behavioral and therapeutic effects via an interac-tion with transport proteins, particularly with SERTs.

In an attempt to obtain a molecular genetic dissection ofthemechanisms regulating the specificity and function ofthe SHTtransport process, we attempted to screen and isolate trans-port proteins from Drosophila. Using a strategy based on thestrong sequence homology between genes encoding membersof the Na+-dependent transporter family (19-22), we reporthere the cloning of a cDNA that encodes a protein, termeddSERT (Drosophila SERT), sharing strong amino acid homol-ogy with the recently cloned human (h) (13-15) and rat (r) (11,12) SERTs.**

MATERIALS AND METHODSClngandE ion ofaDrosophila TransportercDNA. To

screen for potential transporters, an 809-bp BamHI/Sac IcDNA fragment of the human DA transporter, encoding puta-tive transmembrane regions 3-8 (23), was used to probe acDNA library prepared from Drosophila melanogaster heads(6). Briefly, 1.5 x 105 independent clones were lifted in dupli-cate on nylon filters (Colony/Plaque Screen; DuPont) andhybridized as described (23). Filters were washed twice for 30min in 2x standard saline citrate (SSC)/1% SDS buffer at 50°C.After tertiary screening, inserts of seven clones were excisedwith EcoRI, subcloned into pSP73 (Promega), and analyzed byrestriction endonuclease and Southern blot analysis. An EcoRI

Abbreviations: 5HT, serotonin; DA, dopamine; dSERT, Drosophila5HT transporter; GABA, -.aminobutyric acid; hSERT, human 5HTtransporter; NE, norepinephrine; PKA, protein kinase A; PKC,protein kinase C; rSERT, rat 5HT transporter; TM, transmembrane.ITo whom reprint requests should be addressed at: Laboratory ofMolecular Neurobiology, Clarke Institute of Psychiatry, 250 Col-lege Street, Toronto, ON M5T 1R8, Canada.**The sequence reported in this paper has been deposited in theGenBank data base (accession no. U04809).

5158

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

July

27,

202

0

Proc. NatL. Acad. Sci. USA 91 (1994) 5159

3.1-kb fragment (dSERT1), was sequenced in both directionsusing 7-deaza-GTP, Sequenase version 2.0 (United States Bio-chemical), T3/T7 primers (Promega) as well as specific syn-thetic oligonucleotides (Biotechnology Service Centre, Hospi-tal for Sick Children, Toronto) as internal primers.The dSERT 3.1-kb cDNA was subcloned into the mamma-

lian expression vector pcD and, for initial transient expressionstudies, COS-7 cells were transfected with cesium chloride-purified pcDSERT1 by electroporation as described (23).To directly compare the pharmacologic profiles of dSERT

with the previously cloned hSERT (13), the dSERT andhSERT cDNAs were cloned into the plasmids pBluescript IISK+ and pBluescript II KS- (Stratagene), respectively, suchthat initiation methionine codons were downstream of theplasmid-encoded T7 RNA polymerase promoter. To transfectcells for uptake assays, HeLa cells [5 x 104 cells per well in48-well culture plates (Falcon)] were infected with recombi-nant vaccinia virus VTF7.3 (24), which encodes T7 RNApolymerase, at 10 plaque-forming units per cell in Opti-MEMI (GIBCO/BRL)/55 uM 2-mercaptoethanol. The dSERT (100ng per well) or hSERT (50 ng per well) cDNA constructs wereintroduced into the cells by liposome-mediated transfection(Lipofectin; GIBCO/BRL) at a ratio of 1 pg ofDNA/3 pg ofLipofectin mixed in Opti-MEM I/55 ,M 2-mercaptoethanol.[3HJ5HT Uptake Assay. Six hours posttransfection, 5HT

transport assays with 5-hydroxy[G-3H]tryptamine creatininesulfate (PH]5HT; 8.6 Ci/mmol; 1 Ci = 37 GBq; Amersham),100 pM pargyline, and 100 ,uM L-ascorbate in Krebs-Ringersolution/Hepes (KRH) buffer (120mM NaCl/4.7mM KC1/2.2mM CaCl2/1.2 mM MgSO4/1.2 mM KH2PO4/10 mM Hepes,pH 7.4) were performed as described (11, 25). Uptake wasterminated by three washes with ice-cold KRH buffer, cellswere solubilized in 1% SDS, and the level of accumulatedPH]SHT was determined by liquid scintillation spectroscopy.Nonspecific [3H]5HT uptake in HeLa cells was assessed byparallel transfections with the parent plasmid alone, which wassubtracted from the data, or in the presence of 1 ,uM parox-etine for COS-7 cells.Na+ dependence of [3H]5HT uptake was assessed in KRH

buffer with isotonic replacement of NaCl with LiCl, whereasCl- dependence was assessed in KRH buffer with Cl- saltsreplaced by sodium gluconate, potassium gluconate, andCa(NO3)2 at molarities equivalent to regular KRH. SubstrateKm and antagonist Ki values were determined by nonlinearleast-squares fits (Kaleidagraph; Synergy Software, Reading,PA) using either the Hill equation for a rectangular hyperbolaor the four-parameter logistic equation with necessary adjust-ments for substrate concentration (26).Chromosomal and in Situ Localization. In situ hybridization

to polytene chromosomes and embryos was performed usingmethods described by Quan et al. (6).

RESULTS AND DISCUSSION

Homology screening with a fragment of the human DA trans-porter cDNA encoding transmembrane domain (TM)3-TM8was used to identify seven positive clones from a D. melano-gaster head cDNA library. All of these clones were found toencode the same cDNA with the largest ofthe three clones (3.1kb) containg the full-length coding region for dSERT. Nu-cleotide sequence analysis of dSERT revealed consensussequences for translation initiation (27) followed by an openreading frame of 1866 nucleotides encoding a protein of 622amino acid residues with an estimated molecular weight of69,279. Hydropathic analysis of the deduced amino acidsequence of dSERT indicates the presence of 12 putativetransmembrane domains lacking an N-terminal hydrophobicmembrane insertion sequence (28). As such, both the N and Ctermini are constrained within the cytoplasm, suggesting thatthe topological organization ofdSERT is analogous to models

proposed for virtually all members ofthe mammalian Na+/Cl-neurotransmitter cotransporter gene family (19-22). As illus-trated in Fig. 1, amino acid alignment of dSERT with othermembers ofthe sodium-dependent transporter family indicatethat dSERT displays highest overall amino acid sequenceidentity (51%) with the hSERT (13-15) and rSERT (11, 12)sharing 309 identical amino acid residues. dSERT showedslightly lower homology with the human NE (29) and DA (17,18, 23) transporters (47% for both) and has 104 identicallyconserved amino acid residues with all mammalian transport-ers. Substantial sequence divergence between dSERT and theh- and rSERTs is evident, particularly within the amino andcarboxyl tails, some intra- and extracellular loops, and a fewputative transmembrane domains (e.g., TM3 and -12). Inter-estingly enough, dSERT has a conserved aspartic acid residuein TM1 (Asp93), which is found only in mammalian 5HT, NE,and DA transporters. This acidic residue has been suggested(11, 36) to direct the binding of polar amino groups of varioussubstrates and/or inhibitors to monoamine transporters.dSERT contains one putative N-linked glycosylation site

(Asn21) in the second extracellular loop in a position homol-ogous to one of the two sites present in hSERT and rSERT.Moreover, ofthe six consensus sites for phosphorylation, fourare found in putative intracellular regions of dSERT: three inthe N terminus [Ser (PKA; protein kinase A), Thr16, Thr,70(PKC; protein kinase C)] and one PKA site (Ser54) in the loopbetween putative TM10 and TM11 (see Fig. 1). Although fourconserved intracellularPKA and PKC sites are found betweenhSERT and rSERT (13), none of these aligns identically todSERT. A functional role for PKC-mediated effects on the5HT transport process has been documented (37). Whetherthe dSERT uptake process is similarly affected by either PKCor PKA phosphorylation has yet to be determined.An additional structural motif observed in dSERT is the

presence ofa leucine zipper within the second transmembranedomain. This type of motif, suggested to be involved inprotein-protein interactions in several DNA-binding proteins(38), has been observed in other transporters, including thosefor NE, DA, yaminobutyric acid (GABA), and proline. Sur-prisingly, the mammalian SERTs have imperfect leucine zip-pers within this region (see refs. 11, 13, and 22). MammalianDA transporters, however, have an additional leucine zippermotif in TM9 (20, 22), while dSERT contains an imperfectmotifin this region. The structural or functional significance ofleucine zippers in the transport process is currently unknownbut may be involved in transporter dimerization or interactionwith transport-associated modulatory proteins (39).To assess the pharmacologic nature of dSERT, the ability

of cells to mediate uptake ofa number of putative substrates,including [3H]5HT, [3H]DA, and [3H]NE was assessed. Aftertransient transfection in COS-7 cells, only the high-affinitytransport of [3H]5HT was evident. Preliminary data revealedthat [3H]5HT uptake (50-100 nM) was inhibited by 5HT andvarious 5HT uptake compounds such as fluoxetine, parox-etine, and cocaine. Other potential substrates for uptake suchas DA, NE, octopamine, and tyramine did not inhibit[3H]5HT uptake. As shown in Fig. 2A, in transfected HeLacells, [3H]5HT uptake was found to occur with high affinityin a concentration-dependent and saturable manner to asingle class of site, with an estimated Km of 490 + 35 nM anda V.. of 5.16 + 0.12 x 10-18 mol per cell per min (n = 3).The Km of 5HT transport by dSERT is virtually identical tovalues obtained for both the cloned human (463 nM) (see alsoref. 13) and rat (320-529 nM) (11, 12) 5HT transporters.5HT-mediated transport by dSERT displays an absolute

requirement for Na+ ions. As shown in Fig. 2B, substitutionof sodium with lithium decreases dSERT-mediated [3H15HTuptake by =95%, similar to the effects seen for both the clonedh- and rSERTs (12, 13). In marked contrast, however, 5HTuptake by dSERT did not display an absolute requirement for

Pharmacology: Demchyshyn et al.

Dow

nloa

ded

by g

uest

on

July

27,

202

0

5160 Pharmacology: Demchyshyn et al.

dSERThSERTrSERT06A humr.NE humawGARA humenCREATINE rabbiTAURINE dogBETAINE dogPROLINECLYCINE rai

Proc. Natl. Acad. Sci. USA 91 (1994)

RAN SME M

lMDRSCSSDFFAGAAATTGRSNPAPWSDDKESPNNEDDSNEDDGDHTTPAKVTDPLAPKLANNERI LVVSVTERT ET GQKAEF LLAVIq AVMETT PLNSOKLSACEDGEDCOENGVLCKVCPTPGDKVESGQI SNGYSAVPSTGAGDDTRHSI PATTT TLVAELRHGER ETI GKKVDFLLSVIl. YAVDLMETTPLNSQKVLSECKDREDCOENGVLQKGYPTTADRAEPSOI SNGYSAVPSTSAGDEASHSI PAATTTLVAE IROGERIEVTI GKKMDFLLSVI YAVDjL

MSKSKCSVGLMSSVVAPAKEPNAVGPKEVEL LVKECNGVLTSSVTLTNPROSPVEAQDRRETI G#KKIDFLLSVI FAVADVLMLLARMNPCVOPENNGAOTGPEaPLRARKTAELLVVKEERCNGVCLLAPRRDGDAaPRET WGKKI DFLLSVV FAVDIL

IIATrNG5SK VADG S T EVYS EAPVA NOKP KT LV V KVQOKKA A D PD RD TWK GR FD FLMS CV YA IGL,t MK K A E NGI Y SVSGDEMAKKSAENCKYSVSEKKGLCPLIAPGPDCAPAKGDGPAGLGAPGGCLAVPPRET TRQMDFIMSCV FAV lL

MATKEKLCCLKDFHKDILKPSPGKSPGTRPEOEAEGKPPOCREK SSKIDFVLSVA Gf V L,

MIDRKVAVPEDGPPVVSWLPEEGEKLDQEGEDOVKDIRG TNKMEFVLSYA Eli LMKKLOEAHLRKPVTPDLLMTPSDOGDVDLDVDFAAD GN TCGKLDFJLLSCI YCV C L

MAVAHGPVATSSPEONGAVPSEATKKDONLT|RIGN GNCIEFYVLTSV YAVC

AFLVPY CLFL 1FC4 LPL FY EAFLLPY TIMAIFC G IPLFYMAFLLPY TIMAIFC a4IPLFf EAF L VPY L LFFIV IA Ghp L FYA FL PYTLFL IA^G P L FYAFLIPYTFLTL IFA OVPLPFL LVFLIPYVLIALVC IPIFFLEAFLIPYFIFLFGC LIPVFFLEAFFIPYFIFFFTC IPVFFLEAFLVPYFLMLAICIGPLFFLBA F MFIPVYF I MbL V F C IP L FF E

L A LL A LL A LL A LL A LC S LSL

Vl IIV A LL SL

ZJ4ANSMEMBRANE 3

(G FHRC CCLSI WKRIC[PAL V Y.AICLIDIYMGMYYNTIIGWA3VYIGC YHRNICCISI WRKICIPIIFK YAICIIAFVY ASY NTVIMAWALYYIGa YHRNM1G CISI WRNI CIPIFK YAICI IAFYIASYYNTI IAWALVYIG FNR E AAG W K AICPIL V FTVILISLYVGFFF NVIIAWA-LHYIGC YNRE AATV W KKICIPIFFK V YVA ILIALYVGF YNVI IAWSVLVYY

TTSI GLGVWK-LAPF V LAAAVLSFWLNI Y VI ISWAIIYTYFMKA SINV N-IC F YASMSIVFYCNT Y MVLAWG FYYI

a YTSE GITC EKIC LFS YASIVIVALLNIY VI ILAWAITTYY* YTSQ SVTA RKICPI.I LASVVIEAVYiNIYYIIILAWAfLFYLa FSSL PLAY K-ISPLFK A AAMLLI VGLVAIY YNMIIAYVILFYI

FASQ CLGV R-ISPMF V YGMMSVVSTVIGIY wNVVICIAjFVYyIIF

*A+L FA 1FTSK i TS[:ONP TEN C VTSENFTLISSIFTODLPW TSICIKNSIWN TGN CT NYFSEDNITLISISLTODRLPW TSICITNSlWN TGN TNYFAQDNITTLFSSFTTEI. PW HlCNNSiWNSPNC -- SDAHPG DLFSS5F T NP TDCGHT TDPKLL NLYNSFTTT P KCONP TDRC - FSNYSM V

LVKSFTTT P AT CIGHT TPD CVE]FAHEDCANLF QSFSE P A CNHS TP CMEDTMRKN- XLFSSFTSELP T TCTNT TEHCMD --FLNH- SLFASLTSNLP EH CGNW TEHC- LEHRGPKDFVFSMTHVLP ATICINNP 9TPDCIAGVLDASNLTN

dSERT EL-ATSPA KIFFERKVSLESYKGNGCLOFI|hSERT --WTLHSTSPAElE1FYTRHVLO HRSSKGLC DL.GrSESRT - WTLHSTSPAEEEIFYLRHVLaIHCQSKGLDDLL GDA human S SCGD -- SGLN-DTFCGTTPAAEYFERCGVLLHLHQSHGIDDLCNE humn G S V L -GNH- TKYSKYKFTPAAEFYERGVLHLHESSGIHD 1I4GABA hunman N- ----- -* T TNMT SAV V EFWE R N MH - OMT DGL DK PGCREATNIE robbilGSL A - N-LTCDaLAIERRSPV EFWENK.V LSGGLEVPTAURINE dog S L W i - - - TLSTKNFT--- SPVTEFWERNVLS- -LSSGIDDPBETAINE dog GA R T ATSSENFT P E FWE R R V L G- T SGI H DLPROLINEIral GN GCA L P L NL S S .- T V *-.SPSVEEVYWSRYVLHI CGSCGIGRPGLYCINE rat GSRPTAL SGNLSHILFNYTLRTSPSEEYWRLYVLKL -SDD GCDF

TRANSMEMBRANE 4 TERANSMEMBRA

PVKTLAL CYFG FLV V CVWK GVARSA V VTI.APYVVLjIVCI SWOLALCIMLIFTVIYFSIWKcYKTSK V WVATFPYIIALSVTIS WOLTLCIVLIFTVIYFSIWKGVKTSC ATFPYFVLfSVPPRWQLTACLVLVIVLLYFSLWKGVKTSK V T AT MPYVVLTALP CWQLL.ICLMVVVIVLVYFSLWKGVKTS GKY IT TLPYVVLFVOIR WPLAIT LA IAWIL VYFC IWKGVGWT GK]V VYF SATY PYVVSL IIAL 9WEVTLCILLACWVLVYFCYWKGVKSTcGK VYFTATFPYVYLVVSLWFDL.ALCLLLVWLVCFFCIWKGY KSTGKVVYFTATFPFAM LLVALRF ELALCLLLAWLICYFCIWKGVSTTGK]VVYFTATFPYLM LVIEIR NLCLCLLLAWVI VFLCI LKIGVKSSGK V|VYFTIATFPYL LILMEVF LPLLGCLGVSWVVVFILCLIR VKSSVK VIVYF VTIA TFPV LTV

%N E 5

IL L VRJA TAL P q

L L VA!G L P 4AjLLVLR AT LP A

kLL LV GV T LP

A

ILLVFR VTLP AiL FL R G V L P A

VLLVR LTLP AL L R T L P A

LLVR VTLPILFVR VTL A

dSEIRT TPEWHKLKNSK:VWhSERT KPNWOKL LET G VW1I DrsERT KPNWQKLLET V4Sw D

DAhuman SVDFYRLCEASIVW D

NE humen H D F Y R L K E A T DGABA h-man TPNFRKLSDSEV L DCREATlE robblIK P DPW S K L R S P 0 V DTAURINEdog YPDISRLEDP V ID

BETAINE dog KPDOLL.RL.KDP V MDPROLINE ml TPFHHLLSSK V EGlVYCINE rat TPKWDKILEAKVW GD

AN SME MUBR A!I

EAAS FFS~LGPCFAAA 1I4FF SLCGPGFAlkAA aI F FSLGPGFAAT ICF LOGVFAAT 1FIF SYLGACFRAT 14IFFSJYGLGLAGT IFF SYAICLAGT IFFSS ICILAGT IFFSFAICQAAL IFYSLGVGRA AS ]I FY S|L.G C AW

NE 6

TL LA L SS Y N KF NNVLLAFAS5Y N KFNNVLLAFASIY 4 KFNNVLtAFSSYVN4KFTNVLIAFASY K FDNSLIAL SY NSFHNALTAL SYN RFNNAMIVSLGS5 KYKYCLTAL GSY KYHNG L L T F A VS YNTFHGL TMASVYNKFHNN

4rRcMYC

NCS

NC Y

T R A N SUE MB R A N E 7 ' T AN SMEM

A L T S SI 1__T$ FLA VI SVL Y A KT SI D KVG LE- P VR V Y PE E-M GSDALVTSVV C S FVSGFVIFTVL Y AEMRNEDVSEAKDA GPG LFIIYYAEAIANMPASTFFDA L VT SYV N;CMSSF V SGFV IF T VL YMA EUR N E DV S EVAKOA C;P SL L FlTYA EA ANUPAS T vFAIVTTSISSTITNSSLVSFSSF FSFL Y AQKHSVA GU AKD P L Fl YPEAIATL.PLSSAWI VCCSSI CI FVISGFAIFSSIL Y AHEHKVNIEDViATE -qAG LVFiILYVPEAISTL.SGSTVWRDS IIYCCINSC SMFAGFVIFSIV F AHVTKRSIAnV|AAS-I(P LAFILAYPEAVTQLPISPLW

QCMLLGCLC. FSGFAIFSIL F AGEGVDSIADVAES P LAFIAYPKAVTMMPLPTFWSI A L C F LN SAT SF A A F VSVF S L F AC CQ GL P S E VA E S-IP L'ArF A F P KIV6 T M FPL SC WTF VT L S TS L AC FIA F SV Y Sa F L CV P V VA KSA 1P LA FI P QAIM TM L P SPFW

LD S Y! S TN,4C AIT SV Y A G F V I..1 F MAN H L G V D VS FVA D H P G LA F V A Y P ELAL.L 1 L P S P L W

BRANE 8

dAERT SI FF LNL ITLLD ST FGGLEAM ITALCDEYP -RV I GRRREIV5ERT AIIFFL LITIL LDSTFACL EGCV ITIAVLODEFFP --HVWAKRRFrSERT AIIFF LIT CLDSTF AGLEGV ITIAVL lOEF FP*-HIWAKRREDA humnw AVVVFFIMLTLC IDSAMGGhEISV ITGCLIDnEF -- -LLHRHRENE hurrum AVVFFV LLAL LD SSMGG EAV ITGLADFQ - VLKRHRKGABA hu A L F F SMLLMLCDF CTV]EGF *TALVEYP RLLRNRRECREATINErob AA L F FF L L L DOF VGVECGF ITGLLDLLPASYYFRFORETAURINEdog S I L F F I L L LL LDSQFVEVEGCVTSLVDLYPSFLRKGFRREBETAINEdog SCLF F I L F LCLDSOFVCVECLVTASM DMFPSQLRKSGRREPROLINE ral S F L F FF L L L LDSOFAFLETIVTAVT EFPVY.L P- -KKAGLYCINE ral S L L FF F iLL LCGTCFClIl.E TLVY AIVDEVGNEWILa--KKT

TRANSIMEMRAANE .-0

; VLVNFLNVYIGP GLAI LFVVFVEAAGVFWFIV 1VDRFS SD VECMLz4AYVVVKLLEEYAT -GPAVLTYVALIEAVAVVSWFY TCFCRDVKE MLjC4AYVVTLLEEYIAT -GPAVLTVAL EAVAVSWFVYG ITOFCSDV KE M LI!

GjIYVFTLLDH FAA-GTStLFGVL EAIGVAWFYYGVGQFSDDIOOMi;I YV LV L LDT F AA GT S L F AV LME A CGV SWFYG VDR F SNDI CQQM MG.jI YVFKLFDYYSASGMSLLFLVFFECVSI SWFYYVNRFYDNIOGE MVMYVFCQLFDYYSASGTTLLWQAFWECVAVAWVY C ADRFMDDIACMiGMYVFQLFDVYYAASGVCLLWVAFFECFVIAWIYG SDNLYDGI EDMYI FLFDYYACSSGIC.L.FLAMFEVICI SWVY ADRFYDNI E ;IISMYWLV L L DD YSA-SF GL MV V VI T C LA VT R VIYV ICaR F CR0D IHM. LiCWlYW ILLMDNVYAA S FS VYI SC MC V SI M IYGHRNYNFTCD IOLM

TRANSMEMBRAANE i11 TRANSMEMBRANE 12 _

dSERT SK1rP LFWRICI TYIS VFLLTIFIFSI GYKEMLOGEEVYVYP D SYO AVTCSSVLCIPMYIIYKFFFASKGGCRORLQESFCEUNCGSVVPGOOGTSV 622hSERT FS PC14WFWRIC VAISPLFLLFI -CSFLMSPPQLRLF ClNY VY S IL YCGTSSF1C1PTY1AYR-L T PGTTFKERI IKSIT PET PT E PCGDIRLNAV 6 3 GrSERT FSPGlWFWRIC VAISPLFLLFII-CSFLMSPPOLPRLFQNYVW SIiV L tCIGMSSVI (C:PTVYIIYR-L IST PGT L KE R K S T PiET P T E I PCGDIRMNAV 6 3 ODA hu-mm CR P1SVLYWRLC KLVSPCFIL.LFVVVSSI VTFRPP-HYGAY F P ANAL V ATSSMAMS P YAA YKFCSLP-GSFREKLAAIAPIEKDREL VVGEVROFT L RHWL K V

NE humw FR P1:LYWRILC KFVSPAFLLFVVVVSS INFKPLT-YDDYT IF PP ANWV CGIALSSMVLVl-IYVIYKFLST -GSLWERLAVYGITPENEHFL VACRDIRCFCQL.HWLAIGARA hum SRPCIWWKLC SFFTPI IAGGVFIFSAVOMTPLT-MGN YF PK CCGV LMALSSMVL PIGYMAYMFLALK GCSL KQR QCV MV 4PsEDT VR P E N&PE HA OACGS SFTSK A

CREANErabbiY RP C PWMKWC SFFTPLVCMGI F IFNI VYYKPL VYNKTYVYP EAM AFALL SVMLCVPiLHLLGCLLRAKG-CMAESRWOLLTQ PVWGLHH L E RAOQDADVRGLT T LT PTAURINE dog YRP PWMKYS AVVTPVLVGCCCFI FSLVKYVPLTYNKVYVYPT A I GL S L A L SSMMCVPLVMVIRLCQTE -GPF LVRLKYL L T PIRE PNRWAVFR E GYA LOLPPGR R

BETAINEcog YRIPWPLVKtS LFLTPGLLAATFLFSLSQYTPLKYNNIYVY PPW YS F LAL SSMI CVP|L F V T L LKTR GSFKKRLROLTTIP1 5 7 5

PROLINEral FKP GLYFRAC LFLSPATLAALLVYSIVKYOP SEYGSYRFPA AELLLI LMGIL L.SCLMIP,AGMLVAVL SEE -GSLWERLCCQASRIPiAI DWGCPSL F t NRi GMYVAT LA G SCLYCINE ral F PPPLF FQ IC R SPTI FFI FTVIYRPI TYFNH Y AAVA FLMALVV CP VAlFOLCRTDGDTLORlNAKLIS WP0 LI

rTLC YAP TTPI

UA human 6 2 0

NE human 51 a8A8A human Y I a CCREATINE rabbil V SF S S K V V E 6TM 6 3 5TAtJRINE dog SH E T OPHHSRDHDV SSWADGPLPCCL.LTLDSHRTRFTELSICTR VFFF 6 5 6PROLINEral SPKPLMVHMRKYGG I TSFENTAI EVDRE IAEEEEFMSMDOTPPNRRAGRGL PVCPFLGHRG 6 62CLYCINE ral E DGEF VPLPDKAQI P1 VGSNGSLSRLQSRI 6 3 9

FIG. 1. Alignment ofdeduced amino acid sequence ofdSERT with other members of the mammalian Na+-dependent transporter gene family.Boxed and shaded areas denote amino acid residues absolutely conserved (- 16%) among all of the transporters represented. Predictedtransmembrane regions are indicated. Consensus sequences for PKA (e) and PKC (U) are shown. l N-linked glycosylation. Sequence sourcesare as follows: dSERT (this paper), hSERT (13), rSERT (11), DA (23), NE (29), GABA (30), creatine (31), taurine (32), betaine (33), proline(34), and glycine (35).

C1- ions, despite the fact that under identical cell and assay the nitrate concentrations in our assay system (-4 mM) shouldconditions, the uptake of 5HT by hSERT was absolutely be insufficient to activate dSERT-mediated 5HT uptake bydependent on extracellular C1- (see also refs. 12 and 13). The 50%o. This represents direct evidence for the existence of aC1- dependency of hSERT is consistent with SHT transport Cl--facilitated neurotransmitter transporter (41); a fast-studies in native membranes (e.g., refs. 30 and 40), suggesting activating presynaptic reuptake current induced by 5HT ina stoichiometric requirement of one C1- ion per transported neurons of the Hirudo leech appears similarly insensitive tomolecule of5HT (40). As depicted in Fig. 2B, when Cl- was Cl- (10). It would be of extreme interest to determine thereplaced by gluconate, dSERT-mediated SHT uptake was kinetics and molecular basis of the unique ion dependence ofreduced to only -50%9 of control, while 5HT uptake by hSERT 5HT uptake by dSERT and the apparent dissociation of thewas reduced by m99% (13). Increasing Cl- concentrations to Na+/Cl- dependency of the mammalian transport process120 mM facilitated the transport of [3H]5HT by dSERT to during the course of early evolution.control levels. While high concentrations (120 mM) of various [3H]5HT uptake by dSERT was inhibited by numerousanions, such as nitrates, can partially substitute for Cl- (12), SERTs in a concentration-dependent and uniphasic manner

dSERT N1GCChSERT 4NIGOrSERT a ?OG0DA human KOfGNE hunion K4N(;GGABA hum_ KNfG GCCREATINE rabbi( KN G GTAURINE dog KN C QGBETAINE dog KONGGPROLINE rol T NO (GGLYCINE rel RN G GC

3pRA N E i

n ir m" r a v I'.-C~lV RSF PYlI:CY

I6 rVR F P Y ICY

A N V,WR F IjC YVN RFPYLICY

V"I RFPYLjCCCtVi R F P Y:,L C GJV AW FPFYLIC Y

NV RFPTjLCVNVWR:P L|CYNYWFP r SAYr 11 fl.

D E q K Y!Y7IL.

61RG VL

FY L.

10114 IRAIVI.6 Dq ";Y L659N4 INA1 ILK~qILF[VLILD YC L

FTGA MIK F IL

WK QR Y LIFTI: MY YI. L

TR ANSME MB RAAE

ILFVLLLLAF FLCALPTMT;YGRF V L A V V T C F F GS LV T L TFGW FVLIVVITCVLGSLLTLT SGL F T L F V L A T F L S L F C V T NG.LFT FCGVT FST F L LALFCI T K.LFIAAVCIISYLIGLSNI TGIISVALCCALCFVIDLSMVTDG:I F IA FlC S ISYL LGL SMVT EGILLILAIAVFCYLAGLFLVTEVFSGLICVAMYLMGLILTTIDIVVYT L GVA VAGCF L L G PL T S.CA

C rclsil elD0 rf71(4 T

.1.A , , -'j, '. , . -JA"01c,XA

Dow

nloa

ded

by g

uest

on

July

27,

202

0

Proc. Natl. Acad. Sci. USA 91(1994) 5161

4-

xsE

0

0.I-

a

I2

0

1

IE

I

[5HTf, iM

--IU

NaCI Na gluconat. L;Ci pBS SKII(-)

FIG. 2. Pharmacologic characterization of dSERT-mediated[3H]5HT uptake in HeLa cells. (A) Saturation isotherm for [3H]5HTuptake. Cells were incubated with 50 nM [3H]5HT and increasingconcentrations of 5HT for 10 min at 37°C. Nonspecific [3H]5HTuptake was determined by parallel transfections with the parentplasmid lacking the dSERT insert. (Inset) Eadie-Hofstee transfor-mation of the same data. V. = 5.16 x 10-18 mol per cell per min;Km = 490 nM. (B) Na+/Cl- dependence of [3H]5HT uptake bydSERT. Cells were incubated with various salts, which replacedeither Na+ or Cl- at equimolar concentrations, and assayed fordSERT-mediated 5HT transport. Results are representative of threeindependent experiments, each conducted in duplicate.

(as indexed by Hill coefficients close to unity) with a phar-macologic profile consistent with, but not identical to, thehSERT. Various antagonists and substrates inhibited 5HTtransport by dSERT with the following rank order ofpotency:paroxetine mazindol> fluoxetine> citalopram > RTI-55-cocaine - 5HT > desipramine > nomifensine 2 imipramine>> DA/NE/tyramine/octopamine/histamine. K1 values forthese and other compounds are listed in Table 1 along withcorresponding values for these agents at the hSERT. Com-parison of the Ki values of various antidepressants, such asparoxetine and imipramine, for the inhibition of dSERT- andhSERT-mediated SHT uptake reveals that these compoundsdisplay from 3- to 300-fold reduction in affinity for dSERTcompared to hSERT. Possibly, the lack ofCl- ion dependencefor 5HT transport by dSERT may account for the poor affinityexhibited by imipramine (-1 uM) at uptake inhibition, sincethe high-affinity binding of [3H]imipramine to human plateletSERTs has been shown to be Cl- ion sensitive (42). AlthoughWIN 35,428, a structural analogue of cocaine, displayed pooraffinity for dSERT, cocaine was found to inhibit 5HT uptakewith a Ki virtually identical to that of hSERT. Interestinglyenough, RTI-55, an analogue of WIN 35,428 displaying highaffinity for hSERT, was l150-fold less potent at dSERT. GBR12,909, a selective DAergic uptake blocker, did not inhibitdSERT-mediated SHT uptake. In contrast, mazindol, a potentNEergic and DAergic uptake blocker, displayed an unex-pected 30-fold increase in selectivity for dSERT with anestimated Ki of -4 nM compared to -100 nM for hSERT.

Table 1. Ki values for inhibition of [3H]5HT uptake by dSERTand hSERT

Ki, nM dSERT/Compound dSERT hSERT hSERTParoxetine 3.4 ± 1.6 0.25 ± 0.03 13.6Mazindol 3.9 ± 0.02 98 ± 1.7 0.04Fluoxetine 73 ± 5.6 3.0 ± 0.05 24.3Citalopram 88 + 17 4.7 ± 0.2 18.7RTI-55 444 ± 64 3.0 ± 0.06 148Cocaine 464 ± 31 611 ± 66 0.765HT 490 ± 35 463 ± 44 1.06Desipramine 580 ± 147 174 ± 20 3.3Nomifensine 1130 ± 183 840 ± 20 1.35Imipramine 1450 ± 280 4.6 ± 0.9 315GBR 12,909 >10,000 NDWIN 35,428 >10,000 NDDA >10,000 >10,000NE >10,000 >10,000Tyramine >10,000 >10,000Octopamine >10,000 >10,000Histamine >10,000 >10,000Ki values for inhibition of [3H]5HT uptake for various compounds

to HeLa cells transiently transfected with dSERT or hSERT cDNAare listed in accordance with their rank order of potency for dSERT.Data represent means of three independent experiments, each con-ducted in duplicate. ND, not determined.

Potential substrates DA, NE, octopamine, tyramine, andhistamine did not inhibit uptake. These data, when takentogether, clearly suggest that dSERT possibly functions as a5HT transporter in Drosophila.The developmental pattern of dSERT expression was in-

vestigated by in situ hybridization to Drosophila embryos andrevealed the accumulation of dSERT mRNA in a restrictednumber of cell bodies in the ventral ganglion. Accumulation ofdSERT mRNA transcripts in these cells is first apparent atstage 15 (see ref. 43 for staging of embryos) and culminates inthe stereotyped pattern shown in Fig. 3a in all stage 16embryos. dSERT mRNA -appears prior to 5HT receptors,which begin to be expressed at stage 16 and end in stage 17 (5).This embryonic pattern remains unchanged through larval

FIG. 3. Whole-mount in situ localization ofdSERT transcripts inlate stage embryos. (a) Lateral view. (b) Dorsal view. In situhybridization ofdSERTmRNA was performed as described by Quanet al. (6). Strongly staining cell bodies are seen in each neuromere ina pattern similar to that seen for 5HT-containing neurons (3). N,neuropil; SG, subesophageal neuromere; Ti, first thoracic neu-romere; Al and A8, first and eighth abdominal neuromeres. (Bar =100 pm.)

Pharmacology: Demchyshyn et al.

_w

1Mo

Dow

nloa

ded

by g

uest

on

July

27,

202

0

5162 Pharmacology: Demchyshyn et al.

development (data not shown). Positive cell bodies are alsopresent in the brain but their position and number are difficultto determine in whole-mount preparations due to weak andvariable staining. As depicted in Fig. 3, positive cell bodieswere detected in each abdominal (Al-A8), thoracic (T1-T3),and subesophageal (SG1-SG3) ganglion. Cell bodies residejust ventral to the neuropil and are generally clustered to-gether. Starting anteriorly, each subesophageal hemignglioncontained three positive cell bodies, each thoracic hemigan-glion contained two cell bodies, and each abdominal hemigan-glion contained two cell bodies with the exception of A8,which contains a single cell body. This pattern is identical tothat reported for 5HT-containing neurons in the embryonicand larval Drosophila nervous system with the exception thatT1 is reported to contain three 5HT-containing cell bodies (3).In addition, cell bodies expressing dSERT transcripts appearsomewhat earlier (13-15 hr) than SHT-containing cell bodies(16-18 hr). The localization of dSERT mRNA to serotonergicneuronal pathways substantially strengthens the contentionthat dSERT functions as a SHT transporter in Drosophila.The location ofthe gene encoding dSERT was identified by

in situ hybridization to salivary gland chromosomes. Hybrid-ization was present at position 60C on the second chromo-some (data not shown).The availability of the cDNA encoding a SHT transporter

from Drosophila with unique pharmacologic, kinetic, and bio-chemical characteristics may allow, via genetic targeting, forthe assessment of mechanisms by which aberrant 5HT trans-port exerts its effects on the many complex SHT-mediatedphysiological and behavioral events in this organism. Similarly,the availability ofDrosophila mutants defective in the synthesisof5HT [ddc (44, 45)] or other catecholamines [ple (45)] can beused to assess the developmental regulation, expression, andfunction ofthe presynaptic 5HT and other transport processes.Moreover, the generation of interspecies chimeric SERTs,combined with site-directed mutagenesis studies ofmammalianSERTs, may pinpoint those structural motifs and correspondingamino acids that are involved in the maintenance ofhigh-affinitybinding of antidepressants and ion dependence of the 5HTtransport process. In addition, cocaine-mediated inhibition ofvarious invertebrate uptake processes has been suggested tounderlie its action as an insecticidal pesticide (46). The avail-ability of a Drosophila gene encoding a 5HT transporter dis-playing poor capacity to discriminate between cocaine andstructural analogs may allow for the screening of compoundsthat exhibit the potential to act as species-specific insecticideswith reduced abuse potential for vertebrates.

Note Added In Proof. While this manuscript was in press, Corey et al.(47) described the cloning of dSERT cDNA with properties similarto those reported here.

The authors wish to thank F. McConkey, A. Tirpak, and M. Grayfor excellent technical assistance. This work was supported in part bygrants from the National Institutes of Health (DA07390) and theMaflinckrodt Foundation to R.D.B. and from the National Institute onDrug Abuse (DA07223-02) and the Clarke Institute of Psychiatry toH.B.N. L.L.D. is supportedby a Studentshipfom the Ontario MentalHealth Foundation, Z.B.P. is a recipient of the John Cleghorn Fel-lowship from the Canadian Psychiatric Research Foundation, andK.S.S. is supported by a Medical Research Council of CanadaStudentship. H.B.N. is a Career Scientist of the Ontario Ministry ofHealth.

1. Dudai, Y. (1988) Annu. Rev. Neurosci. 11, 537-563.2. Tempel, B., Livingstone, M. & Quinn, W. (1984) Proc. Nat!. Acad.

Sci. USA 81, 3577-3581.3. Valks, A. & White, K. (1988) J. Comp. Neurol. 268, 414-428.4. Witz, P., Amlaiky, N., Plassat, J.-L., Maroteaux, L., Borrelli, E. &

Hen, R. (1990) Proc. Natd. Acad. Sci. USA 87, 8940-8944.5. Saudou, F., Boschert, U., Amlaiky, N., Plassat, J.-L. & Hen, R.

(1992) EMBO J. 11, 7-17.

6. Quan, F., Wolfgang, W. & Forte, M. (1993) Proc. Nat!. Acad. Sci.USA 90, 4236-4240.

7. Vall6s, A. & White, K. (1986) J. Neurosci. 6, 1482-1491.8. Dietzel, I. D. & Gottmann, K. (1988) Dev. Biol. 128, 277-283.9. Wierenga, J. M. & Hollingworth, R. M. (1990) J. Neurochem. 54,

479-489.10. Bruns, D., Engert, F. & Lux, H.-D. (1993) Neuron 10, 559-572.11. Blakely, R. L., Berson, H. E., Fremeau, R. T., Jr., Caron, M. G.,

Peek, M. M., Prince, H. K. & Bradley, C. C. (1991) Nature (Lon-don) 354, 66-70.

12. Hoffman, B. J., Mezey, E. & Brownstein, M. J. (1991) Science 254,579-580.

13. Ramamoorthy, S., Bauman, A. L., Moore, K. R., Han, H., Yang-Feng, T., Chang, A. S., Ganapathy, V. & Blakely, R. D. (1993)Proc. Natl. Acad. Sci. USA 90, 2542-2546.

14. Lesch, K.-P., Wolozin, B. L., Estler, H. C., Murphy, D. L. &Riederer, P. (1993) J. Neural Transm. 91, 67-72.

15. Lesch, K.-P., Wolozin, B. L., Murphy, D. L. & Riederer, P. (1993)J. Neurochem. 60, 2319-2322.

16. Shimada, S., Kitayama, S., Lin, C.-L., Patel, A., Nanthakumar, E.,Gregor, P., Kuhar, M. & Uhl, G. (1991) Science 254, 576-578.

17. Giros, B., El Mestikawy, S., Godinot, N., Zheng, K., Han, H.,Yang-Feng, T. & Caron, M. G. (1992) Mol. Pharmacol. 42, 383-390.

18. Vandenbergh, D. J., Persico, A. M. & Uhl, G. R. (1992) Mol. BrainRes. 15, 161-166.

19. Barker, E. L. & Blakely, R. D. (1994) in Psychopharmacology: 4thGeneration ofProgress, eds. Bloom, F. E. & Kupfer, K. F. (Raven,New York), in press.

20. Giros, B. & Caron, M. G. (1993) Trends Pharmacol. Sci. 14,43-49.21. Amara, S. G. & Kuhar, M. J. (1993) Annu. Rev. Neurosci. 16,

73-93.22. Hoffman, B. J. (1994) in Dopamine Receptors and Transporters:

Pharmacology, Structure, and Function, ed. Niznik, H. B. (Dek-ker, New York), pp. 645-667.

23. Pristupa, Z. B., Wilson, J. M., Hoffman, B. J., Kish, S. J. &Niznik, H. B. (1994) Mol. Pharmacol. 45, 125-135.

24. Fuerst, T. R., Niles, E., Studier, F. W. & Moss, B. (1986) Proc.Nat!. Acad. Sci. USA 83, 8122-8126.

25. Blakely, R. D., Clark, J. A., Rudnick, G. & Amara, S. G. (1991)Anal. Biochem. 194, 302-308.

26. Cheng, Y.-C. & Prussoff, W. H. (1973) Biochem. Pharmacol. 22,3099-3108.

27. Kozak, M. (1986) Cell 44, 283-292.28. von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21.29. Pacholczyk, T., Blakely, R. D. & Amara, S. G. (1991) Nature

(London) 350, 350-354.30. Nelson, P. J. & Rudnick, G. (1982) J. Biol. Chem. 257, 6151-6155.31. Guimbal, C. & Kilimann, M. W. (1993)-1. Biol. Chem. 268, 8418-

8421.32. Uchida, S., Kwon, H. M., Yamauchi, A., Preston, A. S., Marumo,

F. & Handler, J. S. (1992) Proc. Nat. Acad. Sci. USA 89,8230-8234.33. Yamauchi, A., Uchida, S., Kwon, H. M., Preston, A. S., Robey,

R. B., Garcia-Perez, A., Burg, M. B. & Handler, J. S. (1992) J.Biol. Chem. 267, 649-652.

34. Fremeau, R. T., Jr., Caron, M. G. & Blakely, R. D. (1992) Neuron8, 915-926.

35. Smith, K. E., Borden, L. A., Hartig, P. R., Branchek, T. & Wein-shank, R. L. (1992) Neuron 8, 927-935.

36. Kitayama, S., Shimada, S., Xu, H., Markham, L., Donovan, D. M.& Uhl, G. R. (1992) Proc. Natl. Acad. Sci. USA 89, 7782-7785.

37. Myers, C. L., Lazo, J. S. & Pitt, B. R. (1989) Am. J. Physiol. 257,L253-L258.

38. Landschulz, W. H., Johnson, P. F. & McKnight, S. L. (1988)Science 240, 1759-1764.

39. Veyhl, M., Spangenberg, J., Puschel, B., Poppe, R., Dekel, C.,Fritzsch, G., Haase, W. & Koepsell, H. (1993) J. Biol. Chem. 268,25041-25053.

40. Reith, M. E. A., Zimanyi, I. & O'Reilly, C. A. (1989) Biochem.Pharmacol. 38, 2091-2097.

41. Rudnick, G. & Clark, J. (1993) Biochim. Biophys. Acta Bio-ener-getics 144, 249-263.

42. Humphreys, C., Beidler, D. & Rudnick, G. (1991) Biochem. Soc.Trans. 19, 95-98.

43. Campos-Ortega, J. A. & Hartenstein, V. (1985) The EmbryonicDevelopment of Drosophila melanogaster (Springer, New York).

44. Budnik, V., Wu, C.-F. & White, K. (1989) J. Neurosci. 9, 2866-2877.

45. Budnik, V. & White, K. (1987) J. Neurogenet. 4, 309-314.46. Nathanson, J. A., Hunnicutt, E. J., Kantham, L. & Scavone, C.

(1993) Proc. Nat!. Acad. Sci. USA 90, 9645-9648.47. Corey, J. L., Quick, M. W., Davidson, N., Lester, H. A. & Guas-

telia, J. (1994) Proc. Nat!. Acad. Sci. USA 91, 1188-1192.

Proc. Natl. Acad Sci. USA 91 (1994)

Dow

nloa

ded

by g

uest

on

July

27,

202

0