(serotonin) transporter

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Geerlings. A., Lopez-Corcuera, B. and Aragon. C. (2000) FEES Lett. 470.5 1-54 18 Rowe. 1.. Corradi. N.,Malosio. M. L., Taverna. E.. Halban. P.. Meldolesi. J. and Rosa, P. ( 1999) J. Cell Sci. I 12. 1865- I877 19 Geerlings. A.. Nunez. E., Lopez-Corcuera. B. and Aragon. C. (200 I) J. Biol. Chern. 276. 17584- I7590 20 Gyuris. 1.. Golernis. E. A,. Chertkov. H. and Brent. R. (1993) Cell 75,79 1-803 21 Pan. Y., Decker, W. K.. Huq. A. H. M. and Craigen. W. J. ( 1999) Genornics 58,282-290 22 Ishikawa. K.. Nagase. T.. Suyarna. M.. Miyajirna. N..Tanaka, A., Kotani. H.. Nomura. N. and Ohara. 0. (1998) D N A Res. 5, 169- I76 23 Akhrnanova. A., Hoogenraad, C. C.. Drabek K.. Stepanova. T.. Dortland. 6.. Verkerk T.. Vermeulen, W.. Burgering. B. M.. De Zeeuw, C. I., Grosveld. F. and Galjart. N. (2001) Cell 104.923-935 24 Aitken. A. ( I 996) Trends Cell Biol. 6. 34 1-347 25 Xiao. 6.. Srnerdon. S. J, Jones, D. H.. Dodson. G. G., Soneji. Y.. Atken. A. and Garnblin. S. I. (I 995) Nature (London) 376. 188-191 17

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Rarnarnoorthy. S. and Blakely, R D. ( 1999) Science 285, 763-766 Bernstein. E. M. and Quick M. W. ( I 999) J. Biol. Chern. 274.889-895 Qian Y.. Galli. A,. Rarnarnoorthy. S.. Risso. 5.. DeFelice. L. J. and Blakely. R D. ( 1997) J. Neurosci. 17, 45-57 Rarnamoorthy. S.. Giovanetti. E.. Qian. Y. and Blakely. R. D. ( I 998) J. Biol. Chern. 273, 2458-2466 Sato, K.. Adarns. R. Betz. H. and Schloss. P. ( 1995) J. Neurochern. 65, 1967- I973 Huff, R A,. Vaughan. R. A,. Kuhar. M. J. and Uhl. G. R. (I 997) J. Neurochern. 68,225-232 Appanundararn. S.. Schroeter, S. and Blakely. R D. ( 1998) J. Pharmacol. Exp. Ther. 287,744-75 I Sudhof, T. C. (1995) Nature (London) 375,645-653 Naren. A. P., Nelson, D. 1.. Xie. W.. Jovov,6.. Pevsner. J., Bennett, M. K., Benos. D.1.. Quick, M. W. and Kirk K. L. (1997) Nature (London) 390. 302-305 Bezprozvanny. I., Scheller. R. H. and Tsien. R. W. (1995) Nature (London) 378, 623-626 Beckrnan, M. L., Bernstein. E. M. and Quick M. W. ( I 998) J. Neurosci. 18. 6 103-6 I I 2 Deken. S. L.. Beckrnan. M. L.. Boos. L. and Quick M. W. (2000) Nat. Neurosci. 3, 998- I003

Received 6 July200 I

Structure, function and regulation of the 5-hydroxytryptamine (serotonin) transporter S. Horschia', R. Hummerich and P. Schloss Central Institute of Mental Health, Biochemical Laboratory, J5, 68 I59 Mannheim, Germany

Abstract

reversible. Basal uptake levels could be regained 48 h after cessation of the treatment. Secondly, in order to gain insight into the molecular mechanism of substrate transport, potential interactions of S E R T with the noradrenaline transporter ( N E T ) have been investigated. A chimeric concatameric transporter construct consisting of rat S E R T and rat N E T has been cloned and characterized pharmacologically in a heterologous expression system.

T h e serotonin transporter (SERT), which terminates serotonergic neurotransmission by rapid reuptake of 5-hydroxytryptamine (5-HT) back into the nerve terminal was the subject of two studies. First, the regulatory effects of antidepressants on the expression of S E R T was investigated. After chronic exposure of S E R T to antidepressants, substrate transport and antidepressant binding parameters were determined. Long-term exposure of S E R T to a selective 5-HT-re-uptake inhibitor, citalopram, resulted in the down-regulation of the expression level. T h e observed effects depend on drug concentration and exposure time and are

Introduction In the central nervous system, biogenic monoamine neurotransmitters are transported back into the nerve terminal via specific transporter proteins that belong to a family of Na'/CI--dependent transporters. Among these are the 5-hydroxytryptamine (5-HT; serotonin) transporter (SERT) and the noradrenaline (norepinephrine) transporter (NET). T h i s study focuses on S E R T , which plays a crucial role in serotonergic neurotransmission as it controls the concentration of free active neurotransmitter in the synaptic cleft and represents a molecular target for clinically effective antidepressants. Selective serotonin-re-

Key words: noradrenaline. antidepressant uptake. Abbreviations used: 5-HT, 5-hydroxytryptarnine: HEK-SERT. human embryonic kidney 293 cells that stably express the serotonin transporter; HEK-SmycN. SERT-myc-NET construct stably expressed in human embryonic kidney 293 cells: NE, norepinephrine (noradrenaline): NET, norepinephrine transporter; PKC. protein kinase C: SERT. serotonin (5-hydroxytryptamine) transporter; SSRl selective serotonin re-uptake inhibitor. 'To whom correspondence should be addressed (e-mail shonch(n'as200.zi-rnannheirn.de).

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the incubation, medium was changed daily and, in order as to ensure total removal of antidepressants before performing transport or binding measurements, the cells were incubated for 4 h in drug-free medium and were washed twice with buffer prior to the uptake assay or membrane preparation. Exposure of HEK-SERT cells to citalopram for 3 days resulted in a decrease in the maximum velocity of 5 - H T transport ( V,,,,,) without significant change in substrate affinity (K,) as shown in Figure 1. This effect was concentrationdependent, with a maximum reduction of V,,, to 38 f 6 of control, at a citalopram concentration of 500 nM. An analysis of the time-course of this effect revealed that after a 4 h incubation of HEKS E R T cells with 500 nM citalopram, V,,, of 5 - H T transport was reduced to 55 & 3 " b ; after incubation for 24 h, V,,, is reduced to 4 6 k 3 O l 1 which is not significantly different to the effect observed after a 3 day incubation. Exposure of HEK-SERTcells to 500 n M desipramine resulted in a V,,, of 94 f 4 compared with the control.

uptake inhibitors (SSRIs) bind to S E R T and enhance synaptic concentrations by blocking 5 - H T uptake. Although inhibition of uptake is achieved rapidly and efficiently in eitro, improvement of mood usually occurs in an antidepressant therapy only after 2-3 weeks. Considering the possibility that antidepressant drugs may regulate the activity of S E R T by the induction of longterm adaptive changes and thus, the efficiency of the clearance of 5 - H T from the synaptic cleft, the effect of chronic exposure of S E R T to antidepressants was investigated. Rat S E R T , stably expressed in a mammalian cell line, was exposed to the antidepressant drugs citalopram (an SSRI) and desipramine (an inhibitor of the S E T ) . After drug treatment, ['HIS-HT transport and [3H]citalopram binding parameters were determined. Results similar to those presented here for S E R T have been reported for the regulation S E T by desipramine [ 1,2]. These similarities between the two transporters, as well as their high similarity on an amino acid level (60 "(,), prompted the present study with the hypothesis that functional molecular mechanisms and regulational mechanisms by specific inhibitors may be identical. Therefore, a study of possible interactions of S E R T and N E T was performed. A concatameric protein, composed of one S E R T monomer and one N E T monomer, was constructed and transfected in a mammalian cell line. However, before studying regulation by specific inhibitors, a detailed analysis of the structure/function relationships was carried out, along with a pharmacological analysis of several inhibitors.

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5-HT transport efficiency of HEK-SERT after exposure to different concentrations of citalopram Cells were plated into 24-well dishes and incubated for 3 days in normal medium ( 0 )or in the presence of 2 nM (H).20 nM (+), 200 nM (A), and 500 nM (V)citaloptam, respectively, before measuring 5-HT uptake. The following data were obtained: (normal medium) K,,, = 0.73 p M and V, = 8.2 pmollmg of protein per min: (2 nM citaloptam) K,,, = I.OpM and V, = 6.3 pmollmg of protein per min: (20 nM citalopram) K,, = I .47 1tM and V, = 3.9 pmollmg of protein per min: (200 nM citalopram) rC, = I .29 p M and V, = 2.7 pmollmg of protein per min: (500 nM citaloptam) & = 2.76pM and V, = 2.5 pmol/mg of protein per min. Results are expressed as meansfS.E.M.

Results and discussion Regulationof SERT by antidepressants T h e effects of the SSRI citalopram and of the S E T inhibitor desipramine were studied in a heterologous expression system with human embryonic kidney (HEK) 293 cells that stably express the rat S E R T (HEK-SERT). Cells were exposed to different concentrations of citalopram or desipramine [in Dulbecco's modified Eagle's medium foetal bovine serum, supplemented with 10 penicillin (100 U/ml), streptomycin (100 ,ug/ml), and geneticin (G418, 200 ,ug/ml)] for various times before measuring transport rates of ['HISH T uptake [3] or preparing cell membranes for radioligand binding assays [4]. Exposure of the cells to antidepressant concentrations up to 500 nM did not affect cell growth or cell morphology, as assessed microscopically and by counting viable cells after 4 days of incubation. During

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Biochemical Society Transactions (200 I), Volume 29. part 6

This indicates that the observed effect on S E R T activity is specific because a marked reduction was only observed with citalopram treatment. T o evaluate the observed reduction of S E R T activity on a molecular level we analysed the cell surface expression of transporter molecules, by the determination of ['H]citalopram binding to membranes of HEK-SERT cells that were prepared after incubation with the antidepressants. Exposure of HEK-SERT cells to citalopram led to a decrease of the maximal number of binding sites (B,,,),without a significant alteration in ligand binding affinity (K,,). This effect was dosedependent and a maximal reduction of B,,,,to 53 & 9 o o was observed after incubation with 5 0 0 n M citalopram for 3 days. Incubation of H E K - S E R T cells with desipramine, up to a concentration of 200 nM, did not affect SERTactivity. Owing to the low affinity of desipramine to S E R T (K, = 350 nM, [S]), a slight reduction in B,,, was observed (86 f6 0 0 )at 500 nM desipramine. That means that chronic exposure of H E K - S E R T cells to the SSRI citalopram induces a down-regulation of binding sites and, thereby, a reduction of S E R T activity. Comparable findings have been described for other neurotransmitter transporters, which showed a decrease in transport activity accompanied by a reduction in the number of transporter molecules on the cell surface after exposure to transport antagonists : N E T is selectively downregulated after exposure to desipramine [1,2], and the activity of the y-aminobutyric acid transporter, G A T l , is reduced after incubation with the specific inhibitor tiagabine [6]. T h e extent of the antagonist-induced down-regulation is similar to that observed upon activation of protein kinase C (PKC). P K C phosphorylates monoamine-, yaminobutyric acid- and glycine-transporters, and upon the activation of P K C it has been shown that V,,,,, (the maximal transport rate) is reduced by approx. SOn,,, with no significant change in K, (the apparent substrate affinity) of these transporters [7-141. In the case of the dopamine transporter, it has been reported that upon activation of P K C the transporters are rapidly endocytosed from the cell surface and targeted to the endosomal/lysosomal pathway, where they are completely degraded [14]. A different mechanism, however, is hypothesized for S E R T after P K C activation. I t has been shown here that S E R T is translocated to intracellular compartments from where it can be redistributed back to the cellsurface upon activation of phosphatases [15,16]. T h e mechanism by which long-term exposure to


antidepressants regulates S E R T or N E T activity is presently unclear. T h e time course of recovery of ["HIS-HT uptake was measured after incubation of H E K S E R T cells with 500 nM citalopram for 24-72 h in order to investigate any possible reversibility of the citalopram-induced down-regulation of 5 - H T transport. Different wash-out periods were used since there was no significant difference in V,, reduction for these incubation times. Cells were incubated with the drug for 24 or 48 h. Medium was changed to drug-free medium and the cells were cultured for a further 24 or 48 hours, after which [3H]5-HT-uptake measurements were performed. Cells were also exposed to citalopram for 72 h and a fourth set of cells was used as drug-free control. As shown in Figure 2, after a 1 day washout, transport rates were elevated compared with the cells that were exposed to the drug for 72 h, while after 2 days' wash-out the V,,,, values had returned to basal levels. This observation shows that S E R T activity down-regulated by exposure to citalopram can be recovered and it might be an indication for a reversible internalization of transport proteins, implying that endocytosed proteins might be redistributed on the cell surface. T o gain a better insight into molecular mechanisms underlying the effects observed in this study, further analyses on S E R T trafficking and its subcellular distribution after chronic exposure to transport

Recovery of basal 5-HT uptake levels following citalopram-induced V,, reduction HEK-SERT cells were exposed t o 500 nM citalopram for (A) 24 or (B) 48 h. before changing drug-containing medium t o drug-free medium for another 24 (B) or 48 h (A), after which [)H]5-HTuptake measurements were performed One set of cells (C) was exposed t o citalopram for 72 h and another set (D) was kept drug-free as a control. Results are expressed as meansfSEM *P < 0.05. Student's t test

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for functionality and protein expression. Uptake measurements revealed kinetic constants of K , = 1.1 f 0.1 pM and V,,, = 11.2 f 2.0 pmol/mg of protein per min for ['HIS-HT transport and K , = 1.6 f 0 . 3 pM and V,,, = 18.0f 1.2 pmol/mg of protein per min for ['HINE transport. These values are comparable with the constants for the wild-type transporters [20-221. Analysis of the protein by Western blotting using a specific monoclonal mouse antibody against the mycepitope showed a major band with a length of 125 kDa, with no bands visible at the molecular weights of the monomeric transporters. T h e same result was obtained when using antibodies against NET. These findings imply the integrity of the complete HEK-SmycN-construct. In order to investigate the specificity of substrate transport and antidepressant binding to the concatameric protein, we characterized the pharmacological profile of HEK-SmycN for 5 - H T and N E as well as for the antidepressants citalopram (SERT-selective), nisoxetine ( N E T selective) and imipramine (non-selective). All experiments were carried out in comparison with the wild-type transporter proteins. As seen for 5 - H T and N E transport parameters, the inhibition constants obtained for substrate transport inhibition by the antidepressants were comparable with those of wild-type S E R T and N E T . In addition, we did not observe any cross-reactivity, i.e. citalopram did not antagonize N E uptake, nor did nisoxetine block 5-HT transport. Therefore, we conclude that substrate transport mediated by H E K SmycN is specific, implying that 5 - H T is transported into the cell by the S E R T component, and N E via the N E T component, and that S E R T and N E T , although being physically connected in the concatameric construct do not functionally interact. Whether S E R T and N E T act in the concatameric HEK-SmycN-construct as functional monomers or whether they form homomeric dimers by oligomerization of several concatamers, will be the subject of future studies on the H E K SmycN-construct.

antagonists are required, along with a detailed investigation of S E R T protein turnover. Cloning and pharmacological analysis of a concatameric protein of SERT and NET In recent years, different chimeric transporters have been constructed for structure/function analyses. In the case of S E R T , for example, 5 - H T uptake and radioligand binding assays revealed that rat and human S E R T s show different sensitivities to some transporter ligands. Thus, in order to identify the domains or single amino acids of the transporter molecule that are involved specifically in substrate translocation or in antagonist binding, cross-species chimeras between rat and human S E R T s were constructed to track the speciesspecific pharmacologies. In these chimeras, parts of the human transporter were exchanged with the corresponding part of the rat transporter [17,18]. T o obtain insight into the quarternary structure of S E R T , one approach included the functional analysis of concatameric SERTs consisting of two to four S E R T molecules [19]. In order to perform studies on both the structure/function and the quarternary structure, we combined both methods by constructing a chimeric concatamer consisting of S E R T and N E T . Therefore, one molecule rat S E R T and one molecule rat N E T was cloned into pcDNA 3.1. T h e first construct that was made had a linker between the Cterminus of S E R T and the N-terminus of N E T , which consisted of a part of the polylinker of the cloning vector and had a length of nine amino acids. This construct was transfected in H E K 293 cells and tested for substrate transport. Protein expression was analysed by Western blotting. Uptake tests revealed that transport was functional for both substrates, 5 - H T and norepinephrine (NE). However, Western blots using anti-SERT and anti-NET antibodies identified the presence of single transporter molecules at their monomeric molecular weights. That indicates that the construct is not expressed as a dimeric concatamer on the cell surface. Analysis of the mRNAs of the concatamer by reverse transcriptase-PCR and Northern blotting revealed that the SERT-NET concatamer was transcribed as one RNA molecule and thus, we assume, that the concatameric polypeptide is cleaved by proteases in the cytosol before being inserted into the plasma membrane. Therefore, a second construct containing the mycepitope consisting of an 11 amino-acid linker was cloned. This construct was then stably transfected in H E K 293 cells (HEK-SmycN) and was analysed

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Zhu. M.-Y. and Ordway. G. A. ( I 997) J. Neurochem. 68. 134-141 Zhu. M.-Y.. Blakely. R D., Apparsundamm, S. and Ordway, G. A. ( 1998) J. Neurochem. 70, 1547- I555 Sur. C..Betz. H. and Schloss. P. ( I 998) J. Neurochern. 70, 2545-2553 Schloss. P. and Betz. H. ( 1995) Biochemistry 34, 12590-12595

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Stephan. M. M.. Chen. M. A,. Penado. K. M. Y. and Rudnick. G. ( 1997) Biochemistry 36, I 322- I 328 Bemstein. E. M. and Quick. M. W. ( I 999) J. Biol. Chem. 274, 889-895 Qian. Y.. Galli. A,. Ramamoorthy, 8.. Risso. S., DeFelice, L. J. and Blakely. R. D. ( 1997) J. Neurosci. 17, 45-57 Sakai. N..Sasaki. K., Nakashita. M.. Honda, S.. Ikegaki, N. and Saito. N. ( 1997) J. Neurochem. 68,26 18-2624 Osawa. I., Saito, N., Koga. T. and Tanaka. C. (1994) Neurosci. Res. 19, 287-293 Sato. K., Betz. H. and Schloss, P. ( 1995) FEBS Lett 375. 99- I02 Sato. K., Adams, R.. Betz. H. and Schloss. P. ( 1995) I. Neurochem. 65, 1967- I973 Gomeza, J., Zafra. F., Olivares, L.. Gimenez. C. and Aragon. C. ( 1995) Biochim. Biophys. Ada I233,4 I 4 6 Kitayama. 5, Dohi. T. and Uhl. G. R. (1994) Eur. J. Pharmacol. Mol. Pharmacol. 268, I IS- I I 9 Daniels, G. M. and Amara. S. G. ( I 999) J. Biol. Chem. 274, 35794-35801

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Blakely. R D., Ramamoorthy. S., Schroeter, S.. Qian. Y.. Appanundaram. S., Galli. A. and DeFelice. L. J. ( 1998) Biol. Psychiatry 44, 169- I78 16 Ramamoorthy. 8.. Giovanetti. E.. Qian. Y. and Blakely. R. D. ( 1998) J. Biol. Chem. 273. 2458-2466 I 7 Barker. E. L. and Blakely. R D. ( 1996) Mol. Pharmacol. 50. 957-965 I 8 Barker, E. L.. Kimmel. H. L. and Blakely. R D. ( I 994) Mol. Pharmacol. 46, 799-807 I 9 Chang. A. S.. Stames. D. M. and Chang, S. M. (I 998) Biochem. Biophys. Res. Commun. 249.4 I 6 4 2 I 20 Blakely. R D., Benon. H. E.. Fremeau. R. T., Caron. M. G.. Peek M. M.. Prince, H. K. and Bradley, C. C. ( I99 I ) Nature (London) 354,66-70 2 I Hoffman, B. J.. Mezey. E. and Brownstein, M. J. ( I 99 I) Science 254,579-580 22 Pacholczyk T.. Blakely, R. D. and Amara. S. G. ( I 99 I) Nature (London) 350. 350-354 Received 3 May 2001

Impact of oligomerization on the function of the human serotonin transporter J. A. Schmid*. H. Just+ and H. H. Sittei-' *Department of Vascular Biology and Thrombosis Research, Vienna International Research Co-operation Center, Brunnerstrasse 59,A- I234 Vienna, Austria, and +University of Vienna, Austria Institute of Pharmacology, Wahringerstrasse I 3a, A- I090 Vienna, Austria

Abstract

proteins ( C F P and YFP, respectively)]. When expressed in HeLa or HEK-293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were inserted into the plasma membrane and were indistinguishable from wild-type h S E R T on functional testing ( 5 - H T uptake assays, inhibition of 5 - H T uptake by blockers such as imipramine). Oligomers were visualized by fluorescence resonance energy transfer ( F R E T ) microscopy in living cells using complementary methods. Interestingly, oligomerization was not confined to h S E R T ; FRET was also observed between CFPand YFP-labelled rat y-aminobutyric acid transporter. Gel filtration experiments showed that most of the protein was recovered as higher molecular weight complexes; almost no monomeric form was detected. This indicates that the homo-oligomeric form is the favoured state of h S E R T in living cells. T h e formation of oligomers was not significantly affected by co-incubation with transporter substrates or blockers. Based on our observations, oligomer formation might not be essential for the physiological function of the transporter protein, the re-uptake of substrates. Furthermore, we conclude that constitutive oligomer formation might be a general property of Na'/Cl--dependent neurotransmitter transporters.

T h e formation of oligomeric structures has been proposed for a large number of membrane proteins, including G-protein-coupled receptors and ion channels. Biochemical studies employing gel filtration, cross-linking or co-immunoprecipitation techniques showed that the serotonin [5-hydroxytryptamine (5-HT)] transporter is also capable of forming oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. T o test this working hypothesis, we generated fusion proteins of h S E R T and spectral variants of green fluorescent protein [cyan and yellow fluorescent

Key words: amphetamine derivatives. fluorescence resonance energy transfer, green fluorescent protein. membrane protein, uptake inhibiton. Abbreviations used: CFP, cyan fluorescent protein; DAT, dopamine transporter: FRET, fluorescence resonance energy transfer: GFP. green fluorescent protein: GLUT, glucose transporter: GPCR. G-protein-coupled receptor: HEK-293. human embryonic kidney-293 cells: 5-HT. 5-hydroxytryptamine (serotonin): (h)SERT. (human) serotonin transporter; YFP. yellow fluorescent protein. 'To whom correspondence should be addressed (e-mail harald.sitte(cr,univie.ac.at).

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