the first luminal domain of vesicular monoamine transporters

The First Luminal Domain of Vesicular MonoamineTransporters Mediates G-protein-dependentRegulation of Transmitter Uptake*

Received for publication, April 4, 2006, and in revised form, August 10, 2006 Published, JBC Papers in Press, August 22, 2006, DOI 10.1074/jbc.M603204200

Irene Brunk‡, Christian Blex‡, Sivaramakrishna Rachakonda‡, Markus Holtje‡, Sandra Winter‡, Ingrid Pahner‡,Diego J. Walther§, and Gudrun Ahnert-Hilger‡1

From the ‡Functional Cell Biology, Centre for Anatomy, Charite-Universitatsmedizin Berlin, Philippstrasse 12 D-10115 Berlin,Germany and the §Max-Planck-Institute for Molecular Genetics, Ihnesstrasse 73 D-14195 Berlin, Germany

The activity of vesicularmonoamine transporters (VMATs) isdown-regulated by the G-protein �-subunits of Go2 and Gq,but the signaling pathways are not known.We show here thatno such regulation is observed when VMAT1 or VMAT2 areexpressed in Chinese hamster ovary (CHO) cells. However,when the intracellular compartments of VMAT-expressingCHO cells are preloaded with different monoamines, trans-port becomes susceptible to G-protein-dependent regula-tion, with differences between the two transporter isoforms.Epinephrine induces G-protein-mediated inhibition of trans-mitter uptake in CHOVMAT1 cells but prevents inhibitioninduced by dopamine in CHOVMAT2 cells. Epinephrine alsoantagonizes G-protein-mediated inhibition of monoamineuptake by VMAT2 expressing platelets or synaptic vesicles. InCHOVMAT2 cells G-protein-mediated inhibition of monoa-mine uptake can be induced by 5-hydroxytryptamine (sero-tonin) 1B receptor agonists, whereas �1 receptor agonistsmodulate uptake into CHOVMAT1 cells. Accordingly, 5-hy-droxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets andsynaptic vesicles expressing VMAT2. CHO cells expressingVMAT mutants with a shortened first vesicular loop trans-port monoamines. However, no or a reduced G-protein regu-lation of uptake can be initiated. In conclusion, vesicular con-tent is involved in the activation of vesicle associatedG-proteins via a structure sensing the luminal monoaminecontent. The first luminal loop of VMATs may represent aG-protein-coupled receptor that adapts vesicular filling.

Communication between neurons in the central nervous sys-temmainly occurs at specialized structures, the synapses. Vari-ations in the input and output at synapses confer to synapticplasticity, which involves changes at the post- and presynapticsites, respectively. At the presynaptic site, availability andfusion competence of synaptic vesicles as well as the vesicular

transmitter content contribute to the strength of postsynapticanswers.Vesicular monoamine transporters (VMATs)2 translocate

monoamines from the cytosol into the secretory vesicles ofmonoaminergic neurons, neuroendocrine cells, and platelets.Transport is driven by an electrochemical proton gradient(��H�) across the vesicular membrane, which is generated bya vacuolar H�-ATPase (1). Inmammals two closely related iso-forms of the monoamine transporter, termed VMAT1 andVMAT2, respectively, were identified (2, 3). The transporterproteins presumably contain 12 transmembrane domains andare located on different vesicle subtypes (3–5). Both VMATstransport serotonin, dopamine, epinephrine, and norepineph-rine but differ in their substrate preferences and affinities. Incontrast to VMAT2, VMAT1 prefers epinephrine over norepi-nephrine, and the Kd for serotonin uptake is around 1 �M forVMAT1 but below 1 �M for VMAT2. Furthermore, histamineis only transported by VMAT2. The activity of both transport-ers is irreversibly inhibited by reserpine, whereas tetrabenazineexclusively inhibits VMAT2 (6, 7).We have shown previously that monoamine uptake is regu-

lated by heterotrimeric G-proteins that are associated withtransmitter-containing secretory vesicles. The G-protein G�o2inhibits monoamine uptake in either VMAT1- or VMAT2-ex-pressing neurons and neuroendocrine cells (5, 8, 9). A differentG-protein, G�q, blocks serotonin uptake in platelet granulesthat express VMAT2 (10). Interestingly, G-protein-mediatedinhibition is lost in platelets derived from mice lacking periph-eral TPH1 (tryptophan hydroxylase), which do not containendogenous serotonin (11, 12). However, inhibition is restoredwhen the secretory vesicles of TPH1-deficient platelets are pre-loaded with serotonin or norepinephrine (10, 13). These find-ings indicate that the monoamines stored inside the vesiclecontrol the ability of G-proteins to regulate VMAT activity.However, it is not known how the intravesicular serotonin con-centration is sensed and how the signal is transmitted to theG-protein.The present study was undertaken to shed light on the

upstream signaling events involved in the down-regulation ofmonoamine uptake by G-proteins. As an approach, we used

* This work was supported by the Deutsche Forschungsgemeinschaft. Thecosts of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: AG Functional Cell Biology,Centre for Anatomy, Charite-Universitatsmedizin, Philippstr. 12, D-10115Berlin, Germany. Tel.: 49-30-450-528276; Fax: 49-30-450-528912; E-mail:[email protected].

2 The abbreviations used are: VMAT, vesicular monoamine transporter; CHO,Chinese hamster ovary; 5HT1B, 5-hydroxytryptamine 1B; SLO, streptolysinO; PBS, phosphate buffered saline; PIPES, 1,4-piperazinediethanesulfonicacid; GMP-P(NH)P, guanosine 5�-(�;�-imido) triphosphate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 44, pp. 33373–33385, November 3, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

NOVEMBER 3, 2006 • VOLUME 281 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 33373

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VMAT2 Mediates G-protein Coupling

33374 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 44 • NOVEMBER 3, 2006


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CHO cell lines transfected with VMAT1 or VMAT2. Such cellsexpress endogenous G�o2 but are devoid of monoamine trans-porters or monoamine synthesizing and degrading enzymes,allowing for unperturbed external manipulation of monoam-ines. We found that, similar to our previous observations inplatelets, G-protein-mediated regulation is dependent on theloading status of the vesicles, but with differences between thetwo transporter isoforms. Furthermore, deletion mutagenesisof the first intravesicular loop of VMAT1 andVMAT2 revealedthat this domain is essential for themanifestation of G-protein-dependent transport regulation, suggesting that it is the trans-porter itself that operates as an upstream sensor in the signaltransduction to heterotrimeric G-proteins.

EXPERIMENTAL PROCEDURES

Antibodies—A mouse monoclonal antibody against G�o2(clone 101.4 and 101.1) (14) and rabbit polyclonal antibodiesagainst VMAT1 and VMAT2 (5) were kindly provided by R.Jahn (Max-Planck-Institut fur Biophysikalische Chemie,Gottingen, Germany). Rabbit polyclonal antibodies againstVMAT1 and VMAT2were also purchased fromChemicon Int.and Synaptic Systems (Gottingen, Germany), respectively.Horseradish peroxidase-labeled anti-rabbit or anti-mouse IgGwere obtained fromAmershamBiosciences.AlexaRed- orOre-gon Green-labeled anti-rabbit or anti-mouse IgG were pur-chased fromMoBiTec GmbH (Gottingen, Germany).Transmitters and Receptor Ligands—5-Hydroxy-[3H]tryptamine

trifluoroacetate (serotonin; specific activity, 4.33 TBq/mmol)was purchased fromAmersham Biosciences. The monoaminesepinephrine, norepinephrine, dopamine, serotonin, and hista-mine were obtained from Sigma. The 5HT1B receptor antago-nists isamoltan, GR55562, and SB216641; the 5HT1B receptoragonists anpirtoline, CP94253, and CGS12066B; the �1 recep-tor agonist cirazoline; and the �1 receptor antagonist prazosinewere purchased from Tocris.Toxins—Streptolysin O (SLO) from �-hemolytic strepto-

cocci was kindly supplied by U. Weller (Institut Ray-Rocky-Weller, Baden-Baden, Germany).Mice—Wild-type and peripheral tryptophan hydroxylase

knock-out (Tph1�/�) mice were bred as described (11).Construction of Mutant VMAT DNA—Rat VMAT2 DNA

(accession number M97381) was used to create the deletionmutants of the first luminal loop, one with a substitution ofPro42–Val130 corresponding to89aminoacidsby5�Gly residuesand one with a deletion of the variable region Gln61–Thr113 cor-responding to 53 amino acids. The following oligonucleotides

were used: VMAT2-1, 5�-CATGGCCCTGAGCGATCTGG-3�;VMAT2-2, 5�-AATTCGCCGCCGCCGCCGCCAACCACGA-CGGTGAGCAGCATG-3�; VMAT2-3, 5�-GAATTCGATTTC-CGTAGAGTTTTTCTCATGC-3�; VMAT2-4, 5�-GAATTCG-TCCCTTCGGACTGTCCCAG-3�; VMAT2-5, 5�-GAATTC-CAAGTTGGGCTGCTGTTTGCC-3�; and VMAT2-6,5�-CTCGAGTCAGTCACTTTCAGATTCTTC-3�.PCR was performed using Pfu polymerase, and the ampli-

cons from VMAT2-1/VMAT2-2, VMAT2-1/VMAT2-3,VMAT2-4/VMAT2-6, and VMAT2-5/VMAT2-6 were indi-vidually cloned into PCR-script vector from Stratagene. Subse-quently, the VMAT2-1/VMAT2-2 and VMAT2-1/VMAT2-3were subjected to a restriction digestion using HindIII andEcoRI from the respective PCR-script clones and ligated toeither VMAT2-4/VMAT2-6 or VMAT2-5/VMAT2-6 to gen-erate VMAT2�Q61-T113 (short loop, i.e. variable regiondeleted) and VMAT2�P42-V130 (no loop, i.e. loop substitutedby 5� Gly) (see Fig. 6), respectively. VMAT2 cDNA and theloop deletion mutants were subcloned into pCDNA 3.0 intoHindIII and XhoI sites.VMAT1 DNA (accession number M97380) was used to cre-

ate a deletion mutant of the variable region from His62–Val117using the following oligonucleotides: VMAT1-1, 5�-ATCGGA-TCCATGCTCCAGGTTGTTCTG-3�; VMAT1-2, 5�-CTCG-AATTCCAGAGAAGAGTTGCTGTCTTTG-3�; VMAT1-3,5�-CAGAATTCCCAAAAAACAACTGCTTGCAAGG-3�; andVMAT1-4, 5�-ACACTCGAGTTACTCCCCGCTGCTAG-GATC-3�.PCR was performed using Pfu polymerase, and the ampli-

cons from VMAT1-1/VMAT1-2 and VMAT1-3/VMAT1-4were subjected to a restriction digestion using BamHI andEcoRI for VMAT1-1/VMAT1-2 and EcoRI and XhoI forVMAT1-3/VMAT1-4 from the respective PCR-script clonesand then ligated to generate VMAT1�H62–V117. VMAT1cDNA and the loop deletion mutant were subcloned intopCDNA 3.0 into BamHI and XhoI sites.CHO Cell Lines—CHO cells were transfected with wild-type

VMAT-DNA using calcium-phosphate precipitation. The cellswere grown in 35-mmdishes, the culturemediumwas changed4 h prior to transfection to achieve exponential growth at thetime of transfection. Briefly, plasmid was mixed with calciumand then with phosphate (final concentrations: 10 �g/ml plas-mid DNA, 125 mMCaCl2, and 0.75 mMNa2HPO4). After 1 min100 �l of the resulting suspension was added, and cells wereincubated for 24 h at 37 °C with 5% CO2. Then cells were

FIGURE 1. Expression and activity of VMAT1 and VMAT2 in stably transfected CHO cells. A, intact and SLO-permeabilized cells were incubated with [3H]serotonin (40 nM). Unspecific uptake was determined in the presence of 6 �M reserpine. Transfected but nonpermeabilized (intact) CHOVMAT cells exhibit noreserpine-sensitive uptake. Following permeabilization with SLO, only transfected cell lines show a reserpine-sensitive vesicular accumulation of serotoninbetween 2.5 and 3 pmol/mg of protein. B, postnuclear supernatants of VMAT-expressing and native CHO cells were centrifuged at 360,000 � g for 30 min. Theresuspended pellets were subjected to SDS-PAGE and immunoblot analysis. Native and transfected CHO cells express G�o2. VMAT1 and VMAT2 is detectedonly in the respective stably transfected cell lines. C, VMAT1 or VMAT2 is detected by immunofluorescence microscopy in transfected cell lines using isoformspecific antibodies. D, partial co-expression of VMAT1 and VMAT2 with G�o2 in stably transfected cell lines. Confocal laser scanning microscopy reveals apredominant cytoplasmatic localization of VMAT1 and VMAT2 in CHO cells. G�o2 (detected by clone 101.1), in addition, also localizes to the plasma membrane.Scale bar, 30 �m. E, kinetics of serotonin transport by VMAT1 (top panel) and VMAT2 (bottom panel) were analyzed in stably transfected CHO cells following SLOpermeabilization. Uptake was performed for 10 min at 37 °C using KG-ATP buffer containing 40 nM [3H] serotonin and increasing concentrations of unlabeledserotonin. Transport corrected for unspecific uptake in the presence of reserpine saturated at 100 �M. Under these conditions the Km values were in the rangeof 5–15 �M. Higher affinities were observed when the reaction was carried out at 29 °C with apparent Km ranging between 1 and 3 �M (6; data not shown). Eachexperiment was repeated at least twice. The values represent the means of three samples � S.D.




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washed in PBS containing 1 mM EGTA, detached, and seededon 100 mm dishes. In some experiments the LipofectamineTMtransfection reagent (Invitrogen) was used instead according tothe manufacturer’s instructions.To create stable cell lines, geneticin was added 48 h after

the transfection procedure, and the medium was changeddaily. The remaining cells were isolated by limiting dilution,and the clones were analyzed by immunoblot analysis,immunofluorescence microscopy, and serotonin uptake forVMAT expression and activity. The VMAT1-expressingCHO clone (CHOF14.6) used in some of the experimentswas kindly provided by B. Nurnberg (Heinrich-Heine-Uni-versitat, Dusseldorf, Germany).CHO and CHOVMAT1 cells were cultured at 37 °C, 5% CO2

in Dulbecco’s modified Eagle’s medium/Ham’s F-12 mediumsupplementedwith 10% fetal calf serum, 2mM L-glutamine, 100units/ml penicillin, and 100 �g/ml streptomycin. CHO cellsexpressing wild-type or mutant VMAT2 were cultured in thesame medium supplemented with 400 �g/ml geneticin.Immunoblot Analysis—The cells were collected and centri-

fuged for 5 min at 200 � g. All of the following steps werecarried out at 4 °C. After resuspension in 320mM sucrose, 4mMHEPES, pH 7.3, supplemented with protease-inhibitors (pep-statin, leupeptin, and 1 �g/ml aprotinin, 1 mM phenylmethyl-sulfonyl fluoride), the cells were homogenized using a ballhomogenizer (40 cycles; EMBL Germany). The homogenatewas centrifuged for 10 min at 1000 � g, and the postnuclearsupernatant was spun down for 30 min at 360,000 � g. Theresulting pellet was resuspended in sucrose buffer, analyzedfor protein content, and subjected to SDS-PAGE. Aftertransfer to nitrocellulose, the proteins were analyzed usingthe indicated antibodies and the ECL detection system(Amersham Biosciences).Immunofluorescence Microscopy—The cells were grown on

glass coverslips, washed twice with PBS, and fixed in 4% forma-line in 0.1 M phosphate buffer, pH 7.4, for 45 min at room tem-perature. After three rinses with PBS they were incubated withblocking solution containing 5% normal goat serum and 2%bovine serum albumin dissolved in PBS supplemented with0.1% Triton X-100 for 1 h at room temperature. Incubationwith VMAT1, VMAT2, and G�o2 antibodies diluted in block-ing solution was performed at 4 °C overnight. After two rinseswith PBS the cells were incubated in the dark with fluorescencemarker labeled secondary antibody diluted in 2% bovine serumalbumin in PBS supplemented with 0.1% Triton X-100 for 1.5 hat room temperature.After two rinseswith PBS andone rinse in

water, the coverslips were mounted on glass slides for fluores-cence microscopic analysis.Confocal Laser Scanning Microscopy—The localization of

VMAT1, VMAT2, and G�o2 was visualized using a Leica TCSconfocal laser scanning microscope and a 40� oil immersionobjective. Fluorescent dyes (goat anti-rabbit IgG Alexa Fluor488 and goat anti-mouse IgG Alexa Fluor 594, obtained fromMolecular Probes, Eugene, OR) were excited at wavelengths of488 and 543 nm, respectively. Fluorescent signals from thegreen and red channels were collected sequentially using twofilters at 498–535 and 587–666 nm, respectively. The datafrom the two channels weremerged into two-color images (res-olution 1024 � 1024 pixels).Serotonin Uptake into CHOVMAT Cells—The cells were

collected and centrifuged for 5 min at 200 � g. Incubationwith SLO was performed in KG buffer containing 150 mMpotassium glutamate, 20 mM PIPES, 4 mM EGTA, 1 mMMgCl2, 1 mM dithiothreitol, adjusted to pH 7.0 with KOH asdescribed elsewhere. Briefly, the cells were incubated withSLO (about 500 hemolytic units/3–5 � 105 cells) for 10 minon ice. Under this condition SLO monomers bind to thecholesterol of the plasma membrane. After removal ofunbound SLO, oligomerization and pore formation can beinitiated by elevation of the temperature, restricting perme-ability to the plasma membrane (5, 15).Preloading of intracellular compartments with monoam-

ines or receptor ligands was started by adding 500 �l of KGbuffer containing 1 mMATP supplemented with 1 mM ascor-bic acid and 30 �M, 100 �M, 300 �M, or 3 mM of either epi-nephrine, norepinephrine, serotonin, dopamine, or hista-mine or with 500 nM of the indicated receptor ligands.Incubation was performed at 37 °C for 15 min. Preloadingsolution was removed by centrifugation (2 min, 1090 � g,4 °C). The cells were washed in KG buffer and divided intoindividual reaction cups. Uptake was started by resuspend-ing cells in 50 �l of KG buffer containing 1 mM ATP supple-mented with 1 mM ascorbic acid and 40 nM [3H]serotonin.Additives such as the nonhydrolyzable GTP analogue GMP-P(NH)P or reserpine were applied during this step. Incuba-tion was performed for 10 min at 37 °C and stopped byadding 1 ml of ice-cold KG buffer followed by rapid centrif-ugation. The pellets were then lysed in 0.4% Triton X-100 todetermine radioactivity by liquid scintillation counting andprotein content using the bicinchoninic acid method.SerotoninUptake intoBloodPlatelets—Mouse or rat platelets

were prepared as described elsewhere (10). The pelleted plate-

FIGURE 2. GMP-P(NH)P-mediated inhibition of serotonin uptake by VMAT expressing CHO cells following preloading with various monoamines.Permeabilized CHOVMAT1 (top panel) and CHOVMAT2 (bottom panel) cells were incubated with 30, 300, or 3000 �M of the indicated monoamines for 15 minat 37 °C in KG-ATP buffer. The cells were then washed, and uptake of [3H] serotonin was performed with or without 100 �M GMP-P(NH)P as described. ForCHOVMAT1 cells, prior to preloading there is no GMP-P(NH)P-induced inhibition. After preincubation with 300 �M or 3 mM of each monoamine G-proteinactivation inhibited vesicular uptake of serotonin by 20 – 45%. For CHOVMAT2 cells, prior to preloading, there is no GMP-P(NH)P-induced inhibition. G-proteinactivation inhibits serotonin uptake following preincubation with 300 or 3000 �M of norepinephrine, dopamine or serotonin. The values represent the meansof three samples � S.D. The values are obtained from different experiments each repeated twice and run in triplicate with a control (no preloading) andpreloading with one of the indicated monoamines at concentrations from 30 to 3000 �M. GMP-P(NH)P-induced inhibition in control cells represents the meanvalue (n � 12) from four different experiments. Uptake in the absence of GMP-P(NH)P for each monoamine concentration used during preloading was set as100%; the percentage of the respective GMP-P(NH)P-mediated inhibition is shown. Individual experiments for CHOVMAT1 cells preloaded with epinephrineand for CHOVMAT2 cells preloaded with either epinephrine or norepinephrine are inserted. Preloading with monoamines decreased the following serotoninuptake and initiated G-protein-mediated inhibition. Note that there is no inhibition of serotonin uptake in CHOVMAT2 cells following epinephrine preloading.The stars denote significance calculated according to Student�s t test and subjected to an �-adjustment according to Bonferroni-Holm.




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lets were resuspended in Tyrode-HEPES buffer containing 134mMNaCl, 0.34 mMNa2HPO4, 2.9 mMKCl, 12 mMNaHCO3, 20mMHEPES, 5 mM glucose, 1 mMMgCl2, pH 7.4. Preincubationwith receptor ligands or preloading of platelets was performedin Tyrode-HEPES buffer at 37 °C for 30 or 15 min, respectively,before platelets were subjected to the permeabilization anduptake procedure (10).Preparation of Synaptic Vesicles—Synaptic vesicles were pre-

pared from whole rat brain as described (14). The 350,000 � gpellet was resuspended in potassium glutamate buffer by pass-ing it five times each through 23- and 27-gauge needles,respectively. Then synaptic vesicles were divided into indi-vidual reaction cups. Uptake was performed as describedabove but was stopped by the addition of 400 �l of ice-coldKG buffer followed by rapid centrifugation at 440,000 � g for10 min at 4 °C. The pellets were analyzed for radioactivityand protein content.Experimental Design—All of the experiments presentedwere

repeated at least two or three times. Individual uptake experi-ments were performed in triplicate.

RESULTS

G-protein-mediated Inhibition of VMATActivity Is Reconsti-tuted in CHOCells and Depends on Preloading of Internal Stor-age Organelles—For measuring serotonin uptake into VMAT-containing intracellular vesicles, we permeabilized CHO cellstransfected either with VMAT1 (CHOVMAT1) or VMAT2(CHOVMAT2) with SLO. SLO creates stable pores in theplasmamembrane, which allow for direct access of metabolitesto the cell interior while effectively equilibrating all metaboliteand ion gradients between the cytoplasm and the incubationmedium (5, 15).Permeabilized CHOVMAT1 and CHOVMAT2 cells exhib-

ited robust ATP-dependent serotonin uptake that was inhib-ited by reserpine. Some serotonin accumulation was alsoobserved when nontransfected CHO cells were used, but thisprobably represents unspecific adsorption because it was notaffected by reserpine (Fig. 1A). Similarly, no reserpine-sensitivetransport was observed in either transfected or nontransfectedcells when the permeabilization step was omitted (Fig. 1A). Toexclude that the residual transport activity by nonpermeabi-lized cells is due to the presence of the plasmamembrane trans-porter for serotonin, uptakewas alsomonitored in the presenceof the serotonin inhibitor fluoxetine. No change was observed(not shown).Immunoblot and immunofluorescence microscopic analysis

confirmed that transfection resulted in robust expression ofVMAT1 and VMAT2 in the respective cell lines (Fig. 1, B andC). Furthermore, CHO cells contain endogenous G�o2 asshown by a previously characterized monoclonal antibody spe-cific for G�o2 (Fig. 1B). Double immunolabeling experiments ineither CHOVMAT1 or CHOVMAT2 cells revealed a broad co-localization between G�o2 and the respective transporter oninternal membranes (Fig. 1D). Finally we determined uptakekinetics at different serotonin concentrations into SLO perme-abilized CHOVMAT1 or CHOVMAT2 cells (Fig. 1E). Collec-tively, these data show that both CHOVMAT1 and CHO-VMAT2 cell lines display serotonin uptake with properties

typical for VMATs in other systems and furthermore thatVMATs are localized to internal membranes that also containendogenous Go2.

To investigatewhetherVMATactivity is regulated byG-pro-teins, we performed uptake experiments in the presence of thenonhydrolyzable GTP analogue GMP-P(NH)P, which is com-monly used as an effective activator of trimeric GTPases. Asshown in Fig. 2 (first columns) GMP-P(HN)P did not inhibitserotonin uptake by VMAT1 and VMAT2 expressing CHOcells. However, when permeabilized CHOVMAT1 and CHO-VMAT2 cells were preloaded with increasing concentration ofvariousmonoamines (epinephrine, norepinephrine, dopamine,serotonin, and histamine), G-protein-mediated inhibition ofserotonin uptake became apparent (Fig. 2). These findingsresemble our previous observations of the serotonin-depletedplatelets of Tph1�/� mice, in which G-protein-mediated inhi-bition of serotonin uptake was dependent on preloading withserotonin or norepinephrine (10). Monoamine concentrationsrequired for effective preloading of CHOVMAT cells weresomewhat higher than for preloading of platelets (10). The rea-sons for this difference are unclear, but it is conceivable that thisis related to the need of the VMATs to fill endosomal compart-ments that are considerably larger than secretory vesicles.Next, we investigated whether there are differences between

the two transporters with respect to regulation by G-proteins.In CHOVMAT1 cells GMP-P(NH)P-induced inhibition ofserotonin uptake was observed following preincubation witheach of the monoamines (Fig. 2, upper panel). Preincubationwith epinephrine and serotonin was more effective than withother monoamines, but inhibition was highly significant in allcases (Fig. 2, upper panel).In CHOVMAT2 cells monoamine preloading of the internal

compartments also resulted in down-regulation by GMP-P(NH)P of VMAT activity, but with interesting differences.Norepinephrine wasmost effective, whereas for histamine pre-loading and subsequent inhibition of transport was less pro-nounced and required higher histamine concentrations duringpreloading (26% inhibition at a preloading concentration of 3mM; data not shown). Surprisingly, preloading with epineph-rine (even when applied at a concentration of 3 mM) failed toinduce G-protein-mediated inhibition of VMAT2 activity (Fig.2, lower panel), although epinephrine is known to be trans-ported by VMAT2 (6).Epinephrine Antagonizes GMP-P(NH)P-induced Inhibition

of Serotonin Uptake—To further investigate the unexpectedfailure of epinephrine preloading in eliciting transport inhibi-tion, we first checked whether ephinephrine is indeed a sub-strate for VMAT2 in CHOVMAT2 cells. Serotonin uptake wasmeasured in permeabilized CHOVMAT2 cells in the presenceof increasing concentrations of unlabeled epinephrine. Asshown in Fig. 3A (left panel), epinephrine was effectively com-peting with serotonin for uptake.We next investigated which of the phenotypes (activated

or nonactivated G-protein) prevails if the vesicles are pre-loaded with a mix of epinephrine and another monoamine.For this purpose CHOVMAT2 cells were preloaded withnorepinephrine either alone or in combination with a10-fold higher concentration of epinephrine and dopamine,




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respectively (Fig. 3A, right panel).As expected, preloading withnorepinephrine induced G-pro-tein-mediated down-regulation ofserotonin uptake by VMAT2. Thisinhibition was prevented when inaddition epinephrine was added tothe preloading solution. Inhibi-tion, however, prevailed when acombination of norepinephrineand dopamine was used, showingthat the block of G-protein signal-ing is not only specific for epi-nephrine but also that epinephrineis capable of effectively competingwith the signaling of the othermonoamines.A similar competition was ob-

served in normal serotonin-con-taining permeabilized platelets.Here, G-protein activation reducedserotonin uptake by VMAT2 by 40%.This reduction was prevented whenplatelets were preloaded with epi-nephrine, whereas preloading withdopamine rather enhanced the inhib-itory effect ofGMP-P(NH)P (Fig. 3B).A similar pattern was observed whensmall synaptic vesicles isolated frombrain were used. Again, epinephrinepreloading reduced GMP-P(NH)P-mediated inhibition of serotoninuptake, whereas dopamine preload-ing (if any) had the opposite effect(Fig. 3C). Together these data suggestthat epinephrine prevents G-protein-mediateddown-regulationofmonoa-mine uptake, probably by acting bymeans of an intravesicular receptorsite.Monoamine Uptake Is Differen-

tially Regulated by 5HT1B and �1Receptor Agonists Depending on theVMAT Isoform—Together with ourpreviously published data on theregulation of monoamine uptake inplatelets (10), our findings indicatethat G-proteins down-regulatetransmitter uptake by VMATs andthat this effect depends on thedegree of vesicular filling. The ques-tion then arises regarding how thevesicular monoamine content issensed and how the signal is trans-mitted to the G-proteins. The pre-sumed intravesicular monoaminesensors are pharmacologically differ-ent forVMAT1andVMAT2because

FIGURE 3. Influence of epinephrine on G-protein mediated inhibition of serotonin uptake by VMAT2expressing systems. A, left panel, serotonin uptake was performed in absence (set as 100%) or presence ofvarious concentrations of epinephrine (1–100 �M). 100 �M epinephrine completely abolished uptake, indicat-ing that epinephrine is transported by VMAT2 in VMAT2-expressing CHO cells. Right panel, GMP-P(NH)P-me-diated inhibition of serotonin uptake by CHOVMAT2 cells was analyzed following preloading with 100 �M

norepinephrine (norepin., 100 �M norepinephrine plus 1 mM epinephrine (epin.), and 100 �M norepinephrineplus 1 mM dopamine (dopam.). Uptake in the absence of GMP-P(NH)P was set as 100%, the percentage of therespective GMP-P(NH)P-mediated inhibition is shown. Note that epinephrine overcomes the norepinephrine-induced inhibition, whereas the same amount of dopamine had no effect. B and C, GMP-P(NH)P-mediatedinhibition of serotonin uptake into permeabilized rat blood platelets (B) or rat SSV (C ) was compared with orwithout a preloading with epinephrine or dopamine, respectively. Uptake in the absence of GMP-P(NH)P wasset as 100%; the percentage of the respective GMP-P(NH)P-mediated inhibition is shown. Although preloadingwith epinephrine reduced, preloading with dopamine increased inhibition of serotonin uptake followingG-protein activation. The inset in C shows the values for serotonin uptake underlying the blotted GMP-P(NH)P-mediated inhibition. The values represent the means of three samples � S.D.; each experiment was repeatedtwice.




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the antagonistic effect of epinephrine was only observed inVMAT2-expressing systems (synaptic vesicles, platelets, CHO-VMAT2 cells). To shed more light on the nature of this recep-tor, we screened a variety of monoamine receptor agonists fortheir ability to activate the putative sensor and thus substitutefor monoamine preloading (10). These experiments were car-ried out in serotonin-depleted platelets of Tph1�/� mice (11,12). Preincubation of permeabilizedTph1�/� platelets with the5HT1B receptor agonists anpirtoline and CGS12066B mim-icked the effect of monoamine preloading in that serotoninuptake became sensitive to GMP-P(NH)P. The 5HT1B recep-tor agonist CP94253 had almost no effect (Fig. 4A, left panel). Incontrast, the 5HT1B receptor antagonist isamoltan effectivelyprevented the effect of monoamine preloading on transporterdown-regulation (Fig. 4A, right panel). Similarly, isamoltan andother 5HT1B receptor antagonists (GR55562 and SB216641)abolished GMP-P(NH)P-induced inhibition of serotoninuptake in platelets from wild-type mice or from rats (Fig. 4, Band C). Here, preloading is not necessary for the manifestationofG-protein control because the granules are filledwith endog-enous serotonin. In addition, isamoltan overrode G-protein-mediated inhibition of serotonin uptake by rat synaptic vesicles(SV) (Fig. 4D). In BON cells, which expressmainly VMAT1 andless VMAT2, isamoltan only slightly reduced G-protein-medi-ated inhibition of vesicular serotonin uptake, whereas the5HT1B receptor agonist CGS12066B had no effect (Fig. 4E).Taken together a 5HT1B receptor-like structure appears to beinvolved in the regulation of transmitter uptake by heterotri-meric G-proteins into VMAT2-containing vesicles, irrespec-tive of whether this regulation is mediated by G�o2 (SV) or G�q(platelets).Next we analyzed the effect of monoamine receptor agonists

on influencing G-protein regulation of VMAT1 using CHO-VMAT1 cells. As shown in Fig. 5A, the 5HT1B receptor agonistanpirtoline hadno effect. In contrast, preincubationwith the�1receptor agonist cirazoline (500 nM) induced down-regulation.This pattern is exactly opposite to that observed with CHO-VMAT2 cells, where only anpirtoline was effective (Fig. 5A).These data are in accordance with the effect of antagonists on

FIGURE 4. Involvement of a 5HT1B receptor-like structure in G-protein-mediated inhibition of serotonin uptake by VMAT2. A, serotonin uptake ofpermeabilized blood platelets of Tph1�/� mice in the absence or presence ofGMP-P(NH)P was compared in control platelets and after preincubation with50 �M serotonin or 500 nM of the indicated 5HT1B receptor agonists (leftpanel). In a similar experimental design Tph1�/� platelets were analyzedwithout or with preincubation with 50 �M serotonin, 50 �M serotonin plus500 nM of the 5HT1B receptor antagonist isamoltan, or 500 nM of the 5HT1Breceptor agonist CGS12066B (right panel). GMP-P(NH)P-mediated inhibition

of serotonin uptake is induced by preincubating platelets with serotonin orwith the 5HT1B receptor agonists anpirtoline and CGS12066B; CP94253 hasno effect (left panel). Induction of G-protein-mediated inhibition after pre-loading with serotonin can be prevented by application of the 5HT1B recep-tor antagonist isamoltan (right panel). The inset in A shows the values forserotonin uptake underlying the blotted GMP-P(NH)P-mediated inhibition ofthe right diagram. B and C, serotonin uptake of permeabilized blood plateletsof wild-type mice (B) and rats (C ) in the absence or presence of GMP-P(NH)Pwas compared in control platelets and after preincubation with 500 nM of theindicated 5HT1B receptor antagonists. All of the 5HT1B receptor antagonistsabolished inhibition of serotonin uptake by G-proteins. D, preincubation with500 nM isamoltan abolished G-protein-mediated inhibition of serotoninuptake into rat synaptic vesicles. E, serotonin uptake into permeabilized BONcells in the absence or presence of GMP-P(NH)P was compared in controlcells, after preincubation with 500 nM of the 5HT1B receptor agonistCGS12066B and after preincubation with 500 nM of the 5HT1B receptorantagonist isamoltan. G-protein-mediated inhibition of serotonin uptake wasnot affected by CGS12066B but was slightly reduced by isamoltan in thesepreferentially VMAT1-expressing cells. Uptake in the absence of GMP-P(NH)Pwas set as 100%; the percentage of the respective GMP-P(NH)P-mediatedinhibition is shown. The values represent the means of three samples � S.D.Each experiment was repeated twice. The stars denote statistical significanceaccording to Student’s t test.




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FIGURE 5. Regulation of VMAT1 activity by �1 receptor ligands in CHOVMAT1 and PC 12 cells. A, serotonin uptake of permeabilized CHOVMAT1 (left panel)or CHOVMAT2 (right panel) cells in the absence or presence of GMP-P(NH)P was analyzed following preincubation with 500 nM cirazoline (�1 receptor agonist)or with 500 nM anpirtoline (5HT1B receptor agonist). Cirazoline induces G-protein-mediated inhibition of serotonin uptake into CHOVMAT1 but not inCHOVMAT2 cells, whereas the opposite is true for anpirtoline. The insets in A show the values for serotonin uptake underlying the blotted GMP-P(NH)P-mediated inhibition. B, serotonin uptake into permeabilized PC12 cells in the absence or presence of GMP-P(NH)P was compared in control cells and afterpreincubation with 500 nM of each the �1 receptor antagonist prazosine or the 5HT1B receptor antagonist isamoltan. Only prazosine overcomes the G-protein-mediated down-regulation of serotonin uptake into these VMAT1-expressing cells. Uptake in absence of GMP-P(NH)P was set as 100%; the percentage of therespective GMP-P(NH)P-mediated inhibition is shown. The values represent the means of three samples � S.D. Each experiment was repeated twice. The starsindicate statistical significance according to Student’s t test.




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serotonin uptake in PC12 cells that only express VMAT1 (2).Here, only the �1 receptor antagonist prazosine, but not the5HT1B receptor antagonist isamoltan prevented G-protein-mediated down-regulation of serotonin uptake (Fig. 5B).Taken together, these data show that the pharmacological

profile of G-protein activation differs between VMAT1- andVMAT2-expressing cells, even if the analysis was performedagainst the background of an otherwise identical CHO cell line.We therefore hypothesized that the luminal domains of thetransporters themselves function as upstream sensors. Thishypothesis was tested in the following experiments.The First Luminal Loop of VMATMediates Down-regulation

of Monoamine Uptake by G-proteins—To identify the putativereceptor structure in the VMAT proteins, we first comparedthe sequence of the luminal domains of both transporter iso-forms to identify regions in which the two transporters are dif-ferent from each other. VMATs contain large intravesicularloops between transmembrane domains 1 and 2. The aminoacid sequence diverges in the central part of the loop, particu-larly between amino acids Gln61–Thr113 (VMAT2) or aminoacids His62–Val117 (VMAT1), whereas the rest of the predictedintravesicular sequences are almost identical (2, 3). Therefore,we created deletion mutants of both transporters in whicheither the divergent central part or the complete loop wasdeleted and generated stable CHO cell lines expressing theseVMAT mutants. We then tested whether these transportersstill show normal serotonin uptake, and if so, whether down-regulation of monoamine uptake by G-proteins is maintained.For VMAT2, we created stable CHO cell lines expressing

two mutants: VMAT2�P42-V130 (deletion of the entireloop) and VMAT2�Q61-T113 (deletion of the central por-tion of the loop; Fig. 6A). Expression of the mutant VMAT2variants was verified by immunofluorescence microscopicanalysis (Fig. 6B); no change of the intracellular distributionwith respect to wild-type controls was observed. Uptakeassays of permeabilized cells revealed that both mutatedVMAT2 proteins transported monoamines in a reserpine-sensitive manner and with Km values that are indistinguish-able from those observed for the wild-type protein (Fig. 6C).However, when we tested for G-protein-dependent regula-tion, we observed that neither in CHOVMAT2�Q61–T113nor in CHOVMAT2�P42–V130 was serotonin uptakeinhibited by GMP-P(NH)P following preloading with nore-pinephrine (Fig. 6D, left panel). A similar lack of signaling wasobserved when the 5HT1B receptor agonist anpirtoline wasused instead during the preloading phase (Fig. 6D, right panel).

In a parallel approach, we also created a mutant form ofVMAT1 in which the variable region of the first intravesicularloop was deleted, VMAT1�H62–V117. Again, G-protein-me-diated inhibition of serotonin uptake induced by epinephrine orcirazoline was strongly reduced (Fig. 6E). We conclude that thefirst intravesicular loop of VMATs between transmembranedomains 1 and 2 represents a putative receptor-like structure,which senses the intravesicular monoamine concentration andsignals to a trimeric G-protein to down-regulate transportactivity.

DISCUSSION

Using CHO cells permanently expressing VMAT1 andVMAT2, we have shown that monoamines stored inside thevesicles represent the upstream signal that mediates inhibitionof uptake by G-proteins. The vesicular monoamine sensors areencoded by the first intravesicular loop of the transporters thatdiffer in the pharmacological properties between the twoVMAT isoforms. Our data support the view that monoaminestorage vesicles possess a feedback loop regulation that allowsthem to regulate uptake activity based on the degree of filling,and it is conceivable that this pathway is also used to controlvesicular monoamine content by other signaling pathways.Remarkably, this regulation works for both VMAT1 andVMAT2, irrespective of whether the transporters reside ondense core (5, 8–10) or synaptic vesicles (10) or even on endo-somal compartments in CHO cells (Ref. 28 and this paper).The present work also has some more general implications.

First, it reports on one of the rare examples for G-protein-me-diated signal transduction occurring on endomembranes start-ing from the vesicular lumen. Second, G-protein-mediateddown-regulation of monoamine transport is clearly not cou-pled to a heptahelical receptor. Third, to our knowledge it maybe the first example of a G-protein-coupled receptor that is alsoa transmitter transporter.Generally, VMAT activity depends on an electrochemical

gradient, ��H�, driven by the vesicular proton ATPase. Inde-pendent from this basic regulation, variations in quantal sizehave been reported (16). Neurotransmitter content and conse-quently quantal size can be increased by overexpression ofvesicular transporter molecules, as seen for the vesicular ace-tylcholine transporter (17), VMAT2 (18), and the vesicular glu-tamate transporter VGLUT1 (19). Accordingly, deletion ofeitherVMAT2 (20–22) orVGLUT1 (19) reduces vesicular con-tent. In addition, increasing the presynaptic �-aminobutyricacid concentration (23) or the dopamine precursor DOPAC

FIGURE 6. Failure of G-protein-mediated inhibition of serotonin uptake in VMAT mutants lacking the first intravesicular loop. A, schematic drawing ofthe VMAT2 deletion mutants used. The first two transmembrane domains forming the first intravesicular loop are shown. B, CHO cells, CHOVMAT2�Q61–T113and CHOVMAT2�P42–V130 cells were subjected to immunofluorescence microscopic analysis using a rabbit antiserum against VMAT2. Both VMAT2 mutantsare detected by the antibody when expressed in CHO cells. C, the kinetic analysis of CHO, CHOVMAT2�Q61–T113, and CHOVMAT2�P42–V130 expressing cellsperformed for 10 min at 37 °C reveals no difference from wild-type CHOVMAT2. The Km values are around 6 –12 or 1–2 �M when analyzed either for 10 min at37 °C or for 2 min at 29 °C (not shown), respectively. D and E, serotonin uptake into permeabilized CHO cells expressing wild-type or mutant VMAT2 (D) orwild-type or mutant VMAT1 (E ) in the absence or presence of GMP-P(NH)P was compared after preincubation with 1 mM norepinephrine (D, left panel), 500 nM

anpirtoline (D, right panel), 1 mM epinephrine (E, left panel), or 500 nM cirazoline (E, right panel). In contrast to CHO cells expressing wild-type VMAT2, epineph-rine and anpirtoline failed to induce G-protein mediated inhibition of VMAT2 activity. The inset in D shows as an example the values for serotonin uptakeunderlying the blotted GMP-P(NH)P-mediated inhibition following norepinephrine preloading. Deletion of the first intravesicular loop also declined G-protein-mediated inhibition of serotonin uptake by VMAT1 induced by epinephrine or cirazoline. Because of reduced expression transport activity of VMAT1 loopmutants, serotonin uptake was, however, only 25% of that for the wild-type VMAT1. Uptake in the absence of GMP-P(NH)P was set as 100%; the percentage ofthe respective GMP-P(NH)P-mediated inhibition is shown. The values represent the means of three samples � S.D. Each experiment was repeated twice. Thestars indicate statistical significance according to Student’s t test.




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(24) augments the amount of transmitter released. Interest-ingly, reducing the transmitter content does not change theoverall vesicle morphology at least in mammalian neurons (25,26). Collectively, these data indicate that secretory vesicles canvary their transmitter content depending on the physiologicalrequirements. Because even in the central nervous systempostsynaptic receptors are not saturated, a regulation by othermeans than the availability of transmitter and the electrochem-ical gradient is feasible (for review see Refs. 13, 27, and 37).VMATs are not the only vesicular transporters regulated by

heterotrimeric G-proteins. Functional heterotrimeric G-pro-teins have been found on glutamatergic vesicles (9), and theactivity of VGLUTs is regulated by Go2 (14).All of the monoamines with the exception of epinephrine

induceG-protein-mediated inhibition as expected for a generalregulation of the VMATs. Epinephrine is transported by bothisoforms but only effective in regulating uptake by VMAT1. InVMAT2-expressing systems, however, epinephrine overridesinhibition induced by other monoamines. Obviously featuresfor transport and for inducing G-protein-mediated inhibitionof transport or storage have to be distinguished. The abolishingeffect of epinephrine is confirmed for all of the VMAT2-ex-pressing systems investigated, although its physiological role isnot clear so far. In addition VMAT1 and VMAT2 exhibit dif-ferent receptor like recognition domains identified by �1 or5HT1B agonists and antagonists that appear to reside in thefirst luminal domain. So far a direct comparison of sequencesfrom 5HT1B receptor and the first luminal loop of VMAT2failed, however, to coincide.Somemoregeneral considerationsexclude the involvementof a

classical heptahelical receptor. The relation betweenVMAT1 and�1 receptor-like structures and especially between VMAT2 and5HT1B receptor-like structures applies to all of the systems (i.e.platelets regulated by G�q and synaptic vesicles regulated byG�o2). Assuming that a classical receptor initiates G-protein acti-vation, this receptor should be the same for VMAT1 andVMAT2at least in CHO cells. However, the differences between bothVMAT isoforms are also maintained in the otherwise monoam-ine-freeCHOsystem.Collectively thesedata stronglyargue for thetransporters themselvesworkingas theupstreamsignal forG-pro-tein activation. The first luminal domain of VMAT probably rep-resents a receptor-like structure.Deletionof this loopmaypreventthe binding of the vesicular monoamines, thereby impeding thetransductionover themembrane to theG-proteins localizedat thecytoplasmic site of the vesicle.So far a direct proof for vesicular monoamines as activa-

tors of vesicle-associated G-proteins, corresponding to thewell known scenario of agonist-heptahelical receptor activa-tion-G-protein activation, is lacking, and there is no easy wayto obtain such evidence by direct experiments. It is also notclear whether the activated G-protein inhibits (directly orindirectly by using established down-stream signals) VMATactivity in a feedback reaction or whether it changes thestorage capacity of secretory vesicles by other means. Cross-linking experiments or immunoprecipitation using antibod-ies against either G�o2 or VMAT failed to show a directinteraction and were not successful in the past to confirminteractions between classical heptahelical receptors and

their signaling G-proteins. Besides VMAT a putative candi-date being addressed by the activated G-protein may be thecalcium-dependent activator protein of secretion (CAPS1).Deletion of this protein leads to chromaffine granules withreduced catecholamine content, but the underlying molecu-lar mechanism is unclear (29).An epinephrine-sensitive, 5HT1B receptor-like structure may

represent the putative upstream signal forG-protein activation byVMAT2-expressing vesicles irrespective of whether Go2 or Gq istheG-protein involved. Suchpromiscuity is also seen in themouse�3b receptor, which utilizes both Gs and Gi (30). It has to bepointed out that experiments were performed either in permeabi-lized systems (CHO, bloodplatelets) or on isolated SV. Evenwhenusing permeabilized platelets activation of the thromboxane A2receptor did not change vesicular serotonin uptake (10). In addi-tion neither CHO cells (31) nor platelets (12) express 5HT1Breceptors. Collectively, these data exclude the involvement of aclassical heptahelical receptor of the plasmamembrane. Our datamaybebest explainedbyassuming that the first luminaldomainofVMATsmediates G-protein activation.So far only one seven-membrane-spanning receptor resem-

bling a classical G-protein coupled receptor has been found onendomembranes (32). The so-calledKDEL receptor localizes tomembranes of theGolgi apparatus and is involved in retrogradetransport from the Golgi apparatus to the endoplasmic reticu-lum. Whether this receptor possesses the characteristics of aclassical heptahelical receptor and which G-protein is involvedremains unclear so far (33). Interestingly, Go proteins, whichare the least understood with respect to upstream and down-stream signals, appear to be preferentially not regulated by clas-sical heptahelical receptors. For instance, the neuron-specificgrowth-associated protein GAP43 has been described to acti-vate Go (34). Moreover, in Caenorhabditis elegans RGS7 (reg-ulator of G-protein signaling 7) has G�o inhibiting as well aspromoting activity independent from a heptahelical receptor(35). The plasmamembrane-associated amyloid precursor pro-tein involved inAlzheimer disease also appears to be coupled toGo activation (36). Last but not least, Go2 regulates VMAT andVGLUT activity (5, 14, 37). In the present study we describe forthe first time a signal transduction from the luminal site ofsecretory vesicles. Our data suggest that a transporter, such asVMAT, possesses in addition a receptor-like function localizedin its first luminal loop that senses the intravesicular monoa-mine contents. In the case of VGLUT, the situation is probablymore complex because the efficacy of G-protein regulation islinked to the cytoplasmic chloride concentration (14).In summary the first luminal loop of VMAT probably exhibits

receptor-like functions with pharmacological properties differingbetweenthe two isoforms.This loop is involved in theregulationofmonoamine storage by heterotrimeric G-proteins. The cytoplas-mic domains of VMAT mediating G-protein activation and thesteps leading to reduced vesicular uptake probably involving cyto-plasmic proteins have to be identified in the future.

Acknowledgments—We are indebted to Ursel Tofote and ElisabathSchindler for expert technical assistance and to Dr.Michael Bader forvaluable suggestions and the access to the Tph1�/� mice.




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Winter, Ingrid Pahner, Diego J. Walther and Gudrun Ahnert-HilgerIrene Brunk, Christian Blex, Sivaramakrishna Rachakonda, Markus Höltje, Sandra

G-protein-dependent Regulation of Transmitter UptakeThe First Luminal Domain of Vesicular Monoamine Transporters Mediates

doi: 10.1074/jbc.M603204200 originally published online August 22, 20062006, 281:33373-33385.J. Biol. Chem.

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