expression, and electrophysiological characterization - CalTech Authors

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1188-1192, February 1994

Neurobiology

A cocaine-sensitive Dro8ophila serotonin transporter: Cloning, expression, and electrophysiological characterization JANIS L. COREY, MICHAEL W. QUICK, NORMAN DAVIDSON*, HENRY A. LESTER, AND JOHN GUASTELLAt Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125

Contributed by Norman Davidson, October 1, 1993

transporters for all of these substances exist in the Drosophila nervous system. In fact, in vivo uptake mechanisms have been suggested to be responsible for 5HT accumulation in Drosophila mutants lacking the gene encoding dopa decarboxylase (Ddc) (28). We have used a PCR-based cloning strategy to isolate a Drosophila 5HT transporter (termed dSERT1) that is homologous to the mammalian monoamine transporters, as well as to other mammalian members of the Na+/Cl- superfamily. We show that dSERT1 possesses a number of functional properties of this family, including a dependence on sodium and chloride, a high affinity for 5HT, and electrogenicity. We also observe that dSERT1 is cocaine sensitive, possessing an unusual pharmacological profile with properties more similar to mammalian catecholamine transporters than to indoleamine transporters, and has electrogenic properties similar to the mammalian GABA transporteri

A cins e, high-afnity Drosophila ABSTRACT serotonin (5-hydroxytryptamine; SHT) transporter cDNA, denoted dSERT1, was isolated and characterized in oocytes. dSERT1 shows little transport of other monoamnes and is Na+ and Cl- dependent. Sequence anlsis indicates 12 putative tranmbrane domains and strong homologies (%50%) mmi SHT, norepinephrine, and among dSERT1 and ica transporters. Interest y, the ph

propertiesaof dSERT1, iuding sensitivit to antidepressants, are more similar to those of ammalian cateolie transa SST tnsporters. Two-electrode prers than to m nted H-induced, voltagevoIWe-dmp analysis dpendent currents. Clog and characterization of dSERT1 adds ificantly to our knowledge of the diversity of 5HT transporters with regard to pi sequence, pharmacological profile, and permeation properties. One mechanism for the regulation of synaptic activity is through removal of neurotransmitter from the synaptic cleft via high-affinity transporters located on neuronal and glial plasma membranes. By slowing the removal of neurotransmitter from the cleft, inhibition of these transporters can effectively increase the duration of neurotransmitter action on postsynaptic receptors. Recent cloning studies indicate that neurotransmitter transporters consist of two distinct multigene superfamilies. The members of one family are dependent on sodium and chloride for full activity (Na+/Cltransporters) and their substrates include -y-aminobutyric acid (GABA) and glycine (1-7), the monoamine neurotransmitters (8-14), proline (15), taurine (16), betaine (17), and creatine (18). A second superfamily consists of transport proteins that require sodium and potassium for fills activity (Na+/K+ transporters) and includes the transporters for glutamate (19-21) and certain neutral amino acids (22, 23). Although there is no discernible sequence homology between these two superfamilies, they do share several important features: (i) they transport their substrates with high affinity; (ii) their activity is dependent on membrane potential; and (iii) they are predicted to be integral membrane proteins with multiple transmembrane domaifs ranging from 6 to 10 for the Na+/K+ transporters to 12 for the Na+/Cl- transporters. Although the neurotransmitter transporters cloned to date have all been mammalian, histochemical experiments suggest that members of both of these gene superfamilies are present in a variety of phyla, including crustaceans (24), nematodes (25), and insects (26). The fruitfly Drosophila is a particularly useful model for studying neurotransmitter molecular biology, since its nervous system is well-characterized at the anatomical, neurochemical, and genetic levels. Drosophila use GABA, acetylcholine, glutamate, dopamine, and serotonin (5-hydroxytryptamine; 5HT) as neurotransmitters and also may use such diverse substances as histamine, octopamine, and tyramine (reviewed in ref. 27). It is likely that

EXPERIMENTAL PROCEDURES PCR. Degenerate oligonucleotide primers corresponding to two highly conserved amino acid regions near the first (NVWRFPY) and sixth (DAATQIF) transmembrane domains of the rat GABA transporter (1) were designed using Drosophila codon usage. The primers consisted of a 144-fold degenerate upstream (sense) primer corresponding to bases 345-363 of GAT1 with the sequence (5' to 3') GAG CTC GTC GAC AA(TC) GT(GCT) TGG (CA)G(GCTA) TTC CC(CAG) TA and a 48-fold degenerate downstream (antisense) primer corresponding to bases 1008-1026 of GAT1 with the sequence GGG CCC TCT AGA AA(GA) ATC TG(GC) GT(GAC) GC(GA) GC(AG) TC. (The underlined portions of these sequences indicate the addition of 5' restriction sites for cloning purposes.) With these primers, the PCR will generate a 683-bp product from GAT1 mRNA and a 699-bp product from NET1 mRNA. The amplification protocol consisted of 1 min at 940C, 2 min at 650C, and 2 min at 720C for 25-30 cycles. Gel electrophoresis indicated a band of =700 bp, which was subcloned into pBluescript SK(-). Isolion and Characterization of dSERTl cDNA Clone. The 700-bp product was used in a PCR to synthesize a 32P-labeled probe. This probe was used to screen 1 x 106 phage recombinants from the Drosophila head cDNA library in AEXLX (29). After overnight hybridization at 420C [6x SSPE (lx SSPE = 0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA)/35% formamidel, the filters were washed at a final stringency of 500( in 2x standard saline citrate (SSC)/0.1% SDS. Positive clones were rescued and the insert of one of the three positive clones was subcloned in pGEM-7Zf(+) (Promega) for sequencing and in -iitro transcription. Abbreviations: GABA, y-aminobutyric acid; 5HT, serotonin. *To whom reprint requests should be addressed. tPresent address: Acea Pharmaceuticals, 1003 Health Sciences Road West, Irvine, CA 92715. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. U02296).

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

1188

Neurobiology: Corey et al.

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

Xenopus Oocyte Expression and Transport Assays. dSERT1 linearized with Cla I was used as template for an in vitro transcription reaction (Ambion MegaScript kit, Ambion, Austin, TX). To obtain expression levels large enough to measure the transporter-associated currents, we used a previously described PCR-based procedure (30). Methods for oocyte preparation, injection, flux assays, and electrophysiology are described elsewhere (31). Northern Blot Analysis. Twenty micrograms of total RNA from Drosophila head, body, and embryo was separated on a 1% formaldehyde agarose gel and transferred to a nylon membrane (GeneScreenPlus, DuPont/NEN) and probed with a 506-bp fragment of dSERT1 (48 bp of the C terminus and 468 bp of 3' untranslated region). The membrane was hybridized overnight at 420C (50%o formamide/10% dextran sulfate/i M NaCl/1% SDS), washed at 650C for 15 min (0.1x SSC/1% SDS), and exposed to film for 24 h at -70'C.

RESULTS Identification of dSERT1 as a 5HT Transporter. Degenerate oligonucleotides corresponding to conserved portions of putative transmembrane regions 1 and 7 of the Na+/Cl- transporter superfamily were used to amplify aliquots of a Drosophila head cDNA library. An expected product of =700 bp was observed, and DNA sequencing confirmed that this PCR product encoded a member of the Na+/Cl- superfamily. The insert was used to isolate a full-length cDNA from the same library. Three clones were plaque purified under highstringency hybridization conditions and one (clone 39a) was chosen for sequencing and expression characterization. mRNA synthesized from this clone was expressed in Xenopus oocytes and screened for potential substrates (Fig. 1). Oocytes injected with clone 39a mRNA transported [3H]5HT at 5-fold higher levels than uninjected oocytes when incubated for 30 min in 760 nM [3H]5HT. Uptake of [3H]GABA, [3H]glutamate, [3H]histamine, [3H]dopamine, and [3H]norepinephrine was not significantly over the background levels. This indicates that clone 39a encodes a 5HT transporter, which we have designated dSERT1. Sequencing and Molecular Characterization of dSERT1. Sequencing of dSERT1 revealed a 2461-bp insert with an open reading frame of 1743 bases. The proposed initiator ATG, at base 109, occurs within a reasonable match to the consensus translation initiation site (32). The open reading 600

U/)

1- 3H]5HT -T2- [ 3H]GABA

0

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3- [ 4- [ 5- [ 6- [

a) 0 0

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30

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20

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3

4

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FIG. 1. Transport of various neurotransmitters by dSERT1 expressed in Xenopus oocytes. Final concentrations of the listed neurotransmitters were as follows: 760 nM [3H]5HT, 260 nM [3H]GABA, 370 nM [3H]glutamate, 3.8 ,uM [3H]histamine, 800 nM

[3H]dopamine, 1.3 ,&M [3H]norepinephrine. Each point in these

experiments is the

mean

±

SEM of at least four oocytes.

1189

frame encodes a protein of 581 amino acids with a predicted mass of 67 kDa. This sequence does not contain an in-frame stop codon or an alternative initiation codon in the 108-bp 5' untranslated region of dSERT1. It is possible that there is an upstream initiation codon in the complete mRNA and that the present cDNA encodes a functional truncated protein. Hydropathy analysis (33) predicts 12 putative membranespanning domains, indicating a membrane topology similar to that proposed for all members of the Na+/Cl- transporter superfamily (1). There is one potential N-linked glycosylation site, located in the large extracellular loop between the third and fourth transmembrane regions. The primary amino acid sequence predicts four potential protein kinase C phosphorylation consensus sites: two in putative extracellular regions and two in the intracellular N terminus (Thr-3 and Thr-4). Comparison of the predicted amino acid sequence to those of the mammalian 5HT transporters and to the human norepinephrine transporter reveals considerable homology (Fig. 2): 52% and 53% amino acid identity to the rat and human 5HT transporters, respectively (13, 34); 51% identity to NET1 (8); and 48% identity to the rat/bovine dopamine transporters (9-12). The percentage identity is only 39-43% between dSERT1 and the transporters for GABA (1), glycine (5), proline (15), and taurine (16). Comparison of the dSERT1 sequence to sequences in the Genbank data base using the BLAST program revealed no homologous sequences other than members of the Na+/Cl- transporter family. Northern Blot Analysis. A Northern blot revealed a strong signal at -3.3 kb in total RNA from adult Drosophila heads, but no detectable signal with RNA from adult Drosophila bodies or embryos with an 18-h exposure (data not shown). Thus, dSERT1 is predominantly expressed in structures associated with the head, most likely neurons and/or glia of the cephalic nervous system (35). Transport Studies. Oocytes injected with dSERT1 mRNA exhibit an ion-dependent, high-affinity 5HT uptake system that saturates with increasing 5HT concentration (Fig. 3). Eadie-Hofstee analysis (Fig. 3 Inset) demonstrates a Km of 637 ± 100 nM (mean ± SEM of four experiments), which is similar to the Km values determined for the cloned and endogenous mammalian 5HT transporters (13, 14, 34, 36-39). High-affinity transport of SHT by dSERT1 is dependent on extracellular Na+ and Cl-, a hallmark of this transporter family (40). Substitution of Na+ with either Li+ or choline reduced transport by -95%, while substitution of Cl- with nitrate or acetate inhibited uptake by 85-91% (data not shown). Pharmacological Specificity. Drug sensitivity of dSERT1 was assessed by compounds known to inhibit mammalian biogenic amine transport (Table 1). The antidepressants fluoxetine and clomipramine were 6- to 71-fold lower in potency at dSERT1 than at the mammalian 5HT transporters. By contrast, mazindol and nomifensine, which are strong inhibitors of NET1 and DAT1, but are less effective at the mammalian 5HT transporter, were potent inhibitors of dSERT1. Thus, with respect to this group of antidepressants, dSERT1 has a pharmacological profile more similar to the mammalian catecholamine transporters than to the mammalian 5HT transporters. This characteristic is further underscored by the observation that dSERT1 is -5-fold more sensitive to cocaine, a potent inhibitor of mammalian dopamine and norepinephrine uptake, than are the mammalian 5HT transporters. However, the sensitivity of dSERT1 to amphetamine is closer to that observed for the rat and human 5HT transporters than to the human norepinephrine transporter, possibly because amphetamine is a substrate for 5HT transporters. Therefore, the pharmacological profile of dSERT1 represents a mixture of those seen for the mammalian biogenic amine transporters. It is important to note, however, that the specificity of dSERT1 for 5HT as a

1190



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substrate is quite high; unlabeled 5HT is 158- to 479-fold more potent than epinephrine, dopamine, and norepinephrine in reducing [3H}5HT uptake by dSERT1-injected oocytes. This finding corroborates the results of uptake experiments with labeled catecholamines (Fig. 1) and further establishes that dSERT1 is indeed a 5HT transporter rather than a substrateambiguous catecholamine transporter. We searched for additional organic substrates of dSERT1 by examining their ability to decrease transport of 100 nM [3H]5HT. Tryptamine, which is a substrate for the endogenous platelet 5HT transporter (41), was the most effective inhibitor tested in this series, reducing accumulation of [3H]5HT to 27% ± 5% of control values (mean ± SEM) at 200 ,uM. Tryptophan, a precursor of tryptamine and 5HT, did not decrease SHT transport (105% +- 5.4%) at 200 ,uM, nor did the metabolite of 5HT, 5-hydroxyindoleacetic acid (100% ± 13%) at 100 uM. Tyramine, a putative insect neurotransmitter,

523 523

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FIG. 2.

Alignment

of amino

of dSERT1, rat 5HT transporter (SHTT), human SHT transporter (hSERT), and human norepinephrine transporter (NET1). Alignments were based acid

sequences

on a

Dayhoff matrix using the of the Genetics

PILEUP program

Computer Group sequence analysis software package (Madison, WI). Boxes designate identical

or

conservatively substituted amino acids in all transporter sequences. Putative transmembrane domains for dSERT1 are denoted by horizontal

bars with Roman numerals.

reduced uptake to 61% + 7% of control values at 200 pM. Octopamine and histamine, two other putative insect neurotransmitters, had little or no effect on [3H]5HT transport at 200 AM (82% 15% and 95% 15%, respectively). For comparison, nonradioactive 5HT (acting as a diluent) reduced transport of [3H]5HT by 50%o at 100 nM, as expected. of Transport-Assocated CurIon and Voltage rents. By measurements in individual voltage-clamped oocytes, we found that uptake of [3H]5HT is voltage dependent; for example, in one set of measurements over a 5-min time period, uptake increased from 34 16 fmol per oocyte to 90 27 fmol per oocyte between -40 and -80 mV (data not shown). This result prompted us to ask whether dSERT1 is an electrogenic transporter. We used two-electrode voltage-clamp analysis to characterize 5HT-induced currents. When oocytes expressing dSERT1 were exposed to SHT concentrations of 0.1-30 ,uM, distinct inward currents could ±

±

ence

±

±



1191

mV, there was a 2.4-fold increase in the amount of transportassociated current, similar to the voltage dependence of [3H]5HT uptake. When either Na+ or Cl- is eliminated from the superfusion medium, the 5HT-induced currents are too small for systematic current-voltage analysis.

1 20

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2000

3000

4000

5000

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FIG. 3. Kinetics of [3H]5HT uptake. Data for these experiments are representative of four separate experiments and each point is the mean ± SEM of seven determinations. (Inset) Eadie-Hofstee plot of

initial velocity data.

be measured (Fig. 4A). At saturating concentrations of 5HT and membrane holding potentials of -60 or -80 mV, the largest currents induced were =300 nA. The magnitude of the 5HT-induced inward currents remained essentially constant during 5HT application times as long as 5 min (n = 11). The 5HT-induced currents were observed in dSERT1-expressing oocytes from three separate oocyte batches but were not seen in uninjected oocytes (n = 9) or in control oocytes injected with mRNA encoding the rat brain GABA transporter GAT1 (n = 6). Application of 10 ,uM dopamine, norepinephrine, and epinephrine did not evoke measurable currents (data not shown), further indicating the specificity of dSERT1 for 5HT. Fig. 4B shows the current-voltage relationship for the 5HT-induced current and the effect of ion substitution on these currents. For an oocyte superfused with 5 ,uM 5HT in ND96 medium, the 5HT-induced current increased more than linearly between +40 and -100 mV. Between -40 and -80

When expressed in Xenopus oocytes, dSERT1 reconstitutes a high-affinity, cocaine-sensitive, Na+- and Cl--dependent 5HT uptake system. The apparent Km for 5HT of dSERT1 (637 nM) is comparable to the range of values determined for the cloned mammalian 5HT transporters (13, 14, 34) as well as for native mammalian 5HT transporters (36-39). It has a low affinity for norepinephrine, dopamine, and epinephrine and does not transport other neurotransmitters found in the Drosophila nervous system (e.g., glutamate, GABA, dopamine, or histamine). Although the amino acid sequence of dSERT1 shows various degrees of homology with all known members of the Na+/Cl- transporter superfamily, it is most closely related to the mammalian 5HT, norepinephrine, and dopamine transporters. The observation that dSERT1 has about the same degree of structural relatedness to the three mammalian monoamine transporters correlates with its unusual functional properties. Its sensitivity to antidepressants more closely resembles that of NET1 and DAT1 than that of the mammalian 5HT transporter. Similarly, dSERT1 is more sensitive to cocaine than is the mammalian 5HT transporter. By contrast, the sensitivity of dSERT1 to amphetamine is more similar to that of the mammalian 5HT transporters than to that of NET1 and DAT1. Thus, although dSERT1 has a high substrate specificity for 5HT, it exhibits a pharmacological profile that shares features of both mammalian indoleamine and catecholamine transporters. External application of 5HT to dSERT1-injected oocytes induces an inward current that is associated with 5HT uptake. This indicates that the transporter encoded by dSERT1 is electrogenic and that the net charge movement associated with transport is inward. Furthermore, (i) the substrate-induced current does not inactivate, even with extended substrate application times; (ii) the substrateinduced current has an absolute requirement for external Na+; (iii) the substrate-induced current is strongly dependent on external Cl-; (iv) the current-voltage relationship shows

Table 1. Comparison of IC50 values

Norepinephrine

5HT transporters, nM

transporter, nM NET1t

Dopamine

transporter, nM dSERT1 5HTT* hSERTt DAT§ (rat) (human) Inhibitor (human) (rat) (Drosophila) 27 NA 1.4 548 27 Mazindol 118 7.7 44 NA 100 Nomifensine NA 165 NA 5HT >10,000 >10,000 336 140 184 Cocaine 1,000 1,080 NA 6 NA 33 Fluoxetine 450 NA 250 3.9 1,680 2,217 Desipramine NA NA 7.1 NA 2,670 Clomipramine 881 56.1 20,000 3,180 10,800 Amphetamine NA NA NA >1,000 26,000 Epinephrine 316 NA 139 >10,000 42,000 Dopamine NA NA NA >10,000 79,000 Norepinephrine Final [3H]5HT concentrations were 100 nM, 50-100 nM, and 20 nM in determinations for dSERT1, 5HT transporter (5HTT), and human SERT1, respectively. Final [3H]norepinephrine concentration was 20 nM for NET1 and final [3H]dopamine concentration was 15 nM for dopamine transporter (DAT). NA, not applicable. *Hoffman et al. (13). tRamamoorthy et al. (34). tPacholczyk et al. (8). §Giros et al. (12).


1192


A

7. Borowsky, B., Mezey, E. & Hoffman, B. J. (1993) Neuron 10, 851-863. 8. Pacholczyk, T., Blakely, R. D. & Amara, S. G. (1991) Nature

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FiG. 4. Ion and voltage dependence of SHT-induced inward currents in oocytes expressing dSERT1. (A) Transport-associated inward current for an oocyte voltage clamped at a holding potential of -60 mV. The current was evoked with 10 ,AM SHT superfused in ND96 medium for the time indicated by the bar above the trace. (B) Steady-state currents, in the presence and absence of Na+ and Clin the assay medium, plotted as a function of voltage for a second oocyte. The 5HT concentiration was S pM.

greater than linear increase in inward current with membrane potential in the range of 0 to -80 mV. These electrophysiological properties - of dSERT1 are similar to those shown for the mamntalian GABA transporter GAT1 (42). a

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