Báo cáo khoa học: Functionally distinct dopamine and octopamine transporters in the CNS of the cabbage looper moth* potx

Functionally distinct dopamine and octopamine transporters in the CNS of the cabbage looper moth* Pamela Gallant 1,2 , Tabita Malutan 1,2 , Heather McLean 2 , LouAnn Verellen 1 , Stanley Caveney 2 and Cam Donly 1 1 Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, and 2 Department of Biology, The University of Western Ontario, London, Ontario, Canada A cDNA was cloned from the cabbage looper Tricho- plusia ni based on similarity to other cloned dopamine transporters (DATs). The total nucleotide sequence is 3.8 kb in length and contains an open reading frame for a protein of 612 amino acids. The predicted moth DAT protein (TrnDAT) has greatest amino acid sequence identity with Drosophila melanogaster DAT (73%) and Caenorhabdi- tis elegans DAT (51%). TrnDAT shares only 45% amino acid sequence identity with an octopamine transporter (TrnOAT) cloned recently from this moth. The functional properties of TrnDAT and TrnOAT were compared through transient heterologous expression in Sf9 cells. Both transporters have similar transport affinities for DA (K m 2.43 and 2.16 l M , respectively). However, the competitive substrates octopamine and tyramine are more potent blockers of [ 3 H]dopamine (DA) uptake by TrnOAT than by TrnDAT. D -Amphetamine is a strong inhibitor and L -nor- epinephrine a weak inhibitor of both transporters. TrnDAT- mediated DA uptake is approximately 100-fold more sen- sitive to selective blockers of vertebrate transporters of dopamine and norepinephrine, such as nisoxetine, nomi- fensine and dibenzazepine antidepressants, than TrnOAT- mediated DA uptake. TrnOAT is 10-fold less sensitive to cocaine than TrnDAT. None of the 15 monoamine uptake blockers tested was TrnOAT-selective. In situ hybridization shows that TrnDAT and TrnOAT transcripts are expressed by different sets of neurons in caterpillar brain and ventral nerve cord. These results show that the caterpillar CNS contains both a phenolamine transporter and a catechol- amine transporter whereas in the three invertebrates whose genomes have been completely sequenced only a dopamine- selective transporter is found. Keywords: insect; neurotransmitter; transporter; dopamine; octopamine; cocaine. The catecholamine dopamine (DA), the phenolamine octopamine (OA) and the indolamine serotonin (5-HT) influence a variety of behaviors in invertebrates through their actions as neurotransmitters, neurohormones and/or neuromodulators [1,2]. In insects, these monoamines are slow-acting neurotransmitters that act through binding to multiple metabotropic receptors (G-protein coupled recep- tors) on the surfaces of neurons in the CNS and of cells in many peripheral tissues, including flight and visceral muscle. DA acts directly on neurons involved in insect behaviors such as flight [3]. OA modulates neuromuscular activity and responsiveness to sensory input [2]. Insects depleted of DA and OA become lethargic and show reduced levels of aggression [4]. Similarly, 5-HT has been implicated in aggressiveness in crustaceans [5] and responsiveness to olfactory stimuli in honeybees [6]. Following their release, biogenic amines are selectively retrieved at the neural synapse by a family of Na + /Cl – - dependent transport proteins. These proteins are expressed by distinct subsets of neurons in both the mammalian and insect CNS [7,8]. To date, cDNAs encoding neuronal transporters for serotonin (Drosophila [9,10]), dopamine (Drosophila [11]), octopamine and tyramine (Trichoplusia [12]) have been cloned from the insect CNS [8]. The Drosophila dopamine transporter (DrmDAT) has been shown to have functional resemblance to mammalian norepinephrine and dopamine transporters [11]. In the absence of any other related monoamine transporters besides that for 5-HT in the fly genome, it was proposed that DrmDAT represents a common ancestral catechol- amine transporter for the vertebrate NETs and DATs. Similarly, in the other two completely sequenced inverteb- rate genomes (worm and mosquito), homology searches indicate that there are just two sequences in each organism that encode Na + /Cl – -dependent monoamine transporters, Correspondence to C. Donly, Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3. Fax.: + 519 4573997, Tel.: + 519 4571470, E-mail: donlyc@agr.gc.ca Abbreviations:AM, D -amphetamine; DA, dopamine; DAT, dopamine transporter; DBBT, dibromobenztropine; DIG, digoxigenin; E, epinephrine; ET, epinephrine transporter; GUS, a-glucuronidase; 5-HT, serotonin; NE, norepinephrine; NET, norepinephrine transporter; OA, octopamine; OAT, octopamine transporter; ORF, open reading frame; SERT, serotonin transporter; TA, tyramine; TMD, transmembrane domain. *Note: Part of this work has appeared previously in the form of an abstract published online in the Journal of Insect Science (www.insectscience.org). (Received 4 October 2002, revised 3 December 2002, accepted 9 December 2002) Eur. J. Biochem. 270, 664–674 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03417.x one each corresponding to the fruitfly Na + /Cl – -dependent serotonin and dopamine transporters. On the other hand, sequence comparisons using the moth octopamine trans- porter have revealed no counterpart for this third inverteb- rate monoamine transporter in any of the three fully sequenced invertebrate genomes. The functional characterization of the fly DAT showed that this transporter has little affinity for octopamine as a transport substrate [11]. The moth octopamine transporter (TrnOAT), on the other hand, has similar and high affinity for both dopamine and octopamine [12]. This raises several issues: does the moth OAT substitute for DAT in dopami- nergic signaling pathways in the moth CNS, and what is the nature of the mechanism that substitutes for OAT in the octopaminergic pathways in the fly and nematode CNS? Po ¨ rzgen et al. [11] have proposed that a nontransporter- based inactivation of synaptic octopamine, or removal by less selective transporters, may occur in invertebrates lacking an OAT, but this remains to be confirmed. In this study we have cloned and characterized a dopamine-selective transporter (TrnDAT) expressed by moth neurons, and have compared and contrasted its kinetic and pharmacological properties with moth TrnOAT expressed in parallel. We have also confirmed these proteins are produced from distinct genes by characterization of their expression using Northern analysis and in situ hybridiza- tion. The pattern of DAT RNA expression in the caterpillar CNS was shown to differ from that of OAT and thus, unlike the fly and worm CNS, neurons in the moth CNS have high-affinity transporters for the re-uptake of octopamine as well as dopamine. In vitro expression studies show that compared to TrnDAT, the activity of TrnOAT is consid- erably less sensitive to blockers of monoamine uptake in mammals, such as the plant alkaloid cocaine. The mode of action of cocaine in the lepidopteran CNS may not be primarily on octopaminergic neurotransmission, contrary to the mechanism proposed by Nathanson et al.[13]. Materials and methods RT-PCR cloning of TrnDAT Degenerate PCR was performed using single-stranded cDNA from caterpillar heads as template with primers designed from the amino acid sequences of mammalian GABA transporters (GABA1:NVWRFPY) and mamma- lian dopamine transporters (DAT3:KVVWITAT). The PCR mix contained 0.2 m M dNTPs, 2.5 m M MgCl 2 , 2pmolÆmL )1 degenerate primers, 2.5 U Taq DNA Poly- merase (Life Technologies, Burlington, ON, Canada). The PCR conditions were 94 °C for 2 min, followed by 35 cycles of 94 °C for 45 s, 55 °C for 45 s and 72 °Cfor1min, followed by one 5-min hold at 72 °C. RACE-PCR contained 350 l M dNTPs, 0.4 pmolÆmL )1 primers, 0.75 m M MgCl 2 , 1X Expand buffer 3 (contains 2.25 m M MgCl 2 ), and 2.5 U Expand enzyme (Expand Long Tem- plate PCR System, Roche Diagnostics, Laval, QC, Canada). Cycling conditions were 94 °C for 2 min, followed by 10 cycles of 94 °C for 20 s, 62–65 °Cfor30s,and68°C for 3 min, followed by 12 cycles of 94 °C for 20 s, 62–65 °C for 30 s, and 68 °C for 3 min + 20 s increment per cycle, followedbya20-minholdat68°C. The template was double-stranded cDNA made from caterpillar heads ligated to the Stratagene Zap system cloning vector pBK-CMV. Each primer pairing consisted of one DAT-specific primer and one vector primer. Nested reactions used 1–3 lLof product from the first round of PCR as template. PCR to synthesize the complete cDNA was carried out with the Expand Long Template System using single-stranded cDNA from caterpillar heads as template and the products cloned in pGEM-T Easy (Promega Corporation, Madison, WI, USA). Multiple clones were sequenced on both strands using dideoxy chain termination sequencing (Applied Biosystems, Foster City, CA, USA) and the sequences compared to detect potential errors during amplification. Northern analysis Poly A + -enriched mRNA was isolated from T. ni caterpil- lar head, fat body and epidermal tissues using a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Baie d’Urfe ´ , QC, Canada). The mRNA was transferred from a 1% agarose gel containing 6.5% formaldehyde to Hybond N + nylon membrane (Amersham Pharmacia Biotech) as described by Sambrook et al.[14]. A cDNA fragment encompassing the open reading frame (ORF) was labeled with 32 P using Ready-To-Go DNA Labeling Beads (– dCTP) (Amersham Pharmacia Biotech). Unincorporated nucleotides were removed using a NICK column (Amersham Pharmacia Biotech). Labeled probe (2.5 · 10 6 d.p.mÆmL )1 ) was hybridized in QuikHyb Rapid Hybridization Solution (Stratagene, La Jolla, CA, USA) as directed by the supplier and the blot exposed at )70 °Cto Kodak BioMax MS film for 18 h. In situ hybridization A fragment of the TrnDAT cDNA encompassing the putative ORF was cloned in pGEM-T Easy vector and linearized using the restriction enzymes NcoIorNdeI(New England Biolabs, Mississauga, ON, Canada). In vitro transcription was performed using the Riboprobe in vitro Transcription System (Promega Corporation, Madison, WI, USA), following the manufacturer’s directions, using digoxigenin (DIG)- or biotin-labeled ribonucleotides to generate sense and antisense RNA probes. Brains plus ventral nerve cords were dissected intact from late instar cabbage looper caterpillars under NaCl/P i (130 m M NaCl, 70 m M Na 2 HPO 4 ,3m M NaH 2 PO 4 ,pH7.4).Tissueswere fixed in 4% paraformaldehyde in NaCl/P i for 2–3 h and stored at 4 °CinNaCl/P i /Triton (0.3% Triton X-100 in NaCl/P i ) until use. In situ hybridizations were performed essentially as described by Malutan et al.[12]using 175 ngÆmL )1 of labeled RNA for both sense and antisense probes. Transient expression in Sf9 cells The TrnDAT-encoding fragment was isolated from pGEM-T Easy, ligated into the Bac-to-Bac transfer vector pFastBac 1 (Life Technologies, Burlington, ON, Canada) and then transposed to bacmid as directed by the supplier. Sf9 cells (Spodoptera frugiperda cells) were transfected with the recombinant bacmid using CellFectin reagent (Life Ó FEBS 2003 Monoamine transporters in the moth CNS (Eur. J. Biochem. 270) 665 Technologies). Medium containing TrnDAT recombinant baculovirus was harvested 3–4 days after cell transfection and the virus amplified in T25 flasks of Sf9 cells at 50% confluency. The viral titers in tertiary amplifications were estimated by plaque assay. TrnOAT recombinant baculo- virus was amplified in the same way [12]. Transport assays Assays were performed essentially as described in Malutan et al. [12]. Sf9 cells were infected with recombinant baculo- virus at a multiplicity of infection (MOI) of 0.5 and then incubated at 27 °C for 48 h postinfection. The cells were washedfor1hwithNa + -containing saline (11.2 m M MgCl 2 , 11.2 m M MgSO 4 , 53.5 m M NaCl, 7.3 m M NaH 2 PO 4 ,55 m M KCl, 76.8 m M sucrose, pH to 7.0 with KOH) and then incubated for 3 min in 500 lL saline to which [ 3 H]DA (specific activity 28.0 or 36.1 CiÆmmol )1 ; NEN Life Science Products, Boston, MA, USA) was added. DA uptake was found to be linear for about 6 min (data not shown). Salines contained 5 lLof1 mCiÆmL )1 [ 3 H]DAaddedtosalineonice to give a final concentration of 0.3 l M [ 3 H]DA (except for the two lowest concentrations used in the kinetic analysis). Solutions were warmed to 27 °C immediately before use. DA uptake was terminated by washing the cells several times in Na + -free saline (11.2 m M MgCl 2 , 11.2 m M MgSO 4 , 53.5 m M choline chloride, 7.3 m M KH 2 PO 4 ,55m M KCl, 76.8 m M sucrose, pH to 7.0 with KOH). The wells were then air dried and the radiolabel accumulated by the cells extracted in 500 lL 70% ethanol for 20 min. An aliquot of the extract was added to Ready Safe scintillation fluid (Beckman Coulter, Fullerton, CA, USA) and counted in a Beckman LS 5801 scintillation counter. The affinity of TrnDAT and TrnOAT for DA was determined by measur- ing [ 3 H]DA accumulation at 12 DA concentrations from 0.1 to 20 l M DA. Cells exposed to 0.1, 0.2 and 0.3 l M DA were incubated in saline containing [ 3 H]DA only (1.67, 3.33 and 5 lLof1mCiÆmL )1 [ 3 H]DA). [ 3 H]DA was supplemented with unlabeled DA to give the higher DA concentrations. The affinities of the two transporters for DA (K DA m )and maximum rates of DA uptake (V max ) by virally infected cells were estimated through Eadie–Hofstee transformation of the [ 3 H]DA uptake data. Cells incubated in Na + -free saline or infected with a a-glucuronidase (GUS) recombinant virus were used to correct the experimental data for nonspecific (background) accumulation of [ 3 H]DA. The Na + and Cl – dependence of DA uptake were assessed using salines in which Na + was replaced by equimolar choline + ,Li + ,K + , or NMG + or in which Cl – was replaced by equimolar gluconate – ,I – ,Br – ,NO 3 – ,PO 4 – or HCO 3 – . The ability of DA, OA, TA, NE and D -amphetamine (AM) (all presumed competitive substrates for monoamine uptake in insects) to reduce the uptake of [ 3 H]DA by TrnDAT- and TrnOAT- expressing cells was tested over the concentration ranges shown in Fig. 7. Inhibition data are given as a percentage of uptake in saline lacking inhibitor. The concentration at which each compound reduced DA uptake by 50% (IC 50 value) was determined by Hill analysis (where inhibitor concentration is plotted against I/I max – I on a double logarithmic plot, with I ¼ inhibition and I max ¼ maximum inhibition). The TrnDAT and TrnOAT inhibition constants (K i ) for each competitive inhibitor were derived from the IC 50 values using the Cheng and Prusoff [15] equation, IC 50 ¼ K i ð1 þ½S=k DA m Þ,where[S]isthe[ 3 H]DA concentra- tion used in the experiments. Data given are the mean values ± SD obtained from at least three sets of Sf9 cells infected in parallel with TrnDAT or TrnOAT recombinant virus. Fifteen drugs known to block monoamine uptake in mammals were examined for their ability to reduce [ 3 H]DA uptake by cells expressing TrnDAT or TrnOAT. The cells were exposed to inhibitor alone for 4 min before incubation with [ 3 H]DA plus inhibitor for 3 min. As above, each drug was tested at 12 different concentrations on three or more parallel cultures of infected Sf9 cells. [ 3 H]DA accumulation was normalized to DA uptake by untreated cells after correcting for the radioactivity associated with cells exposed to [ 3 H]DA in Na + -free saline. IC 50 values for each drug were determined by nonlinear regression analysis of Hill plots using MICROSOFT EXCEL 2000. The drugs used were benzo[b]thien-2-yl-N-cyclopropylmethylcyclohexan- amine fumarate, {1-[1-(2-benzo[b]thienyl)cyclohexyl]}pipe- ridene maleate, desipramine hydrochloride, imipramine hydrochloride, maprotiline hydrochloride, 3a-[(4-chloro- phenyl)phenylmethoxy]tropane hydrochloride, GBR12909 dihydrochloride from Tocris-Cookson (Ballwin, MO, USA) and amfonelic acid, D -amphetamine, bupropion hydrochloride, N-(2-chloroethyl)-N-ethyl-2-bromobenzyl- amine hydrochloride (DSP-4), clomipramine hydrochloride, cocaine, fluoxetine hydrochloride, nisoxetine hydrochloride, nomifensine maleate and xylamine hydrochloride from Sigma/RBI (St Louis, MO, USA). 4¢,4¢-Dibromobenztro- pine hydrochloride was a gift from the US NIMH Synthesis Program. Results Characterization of a caterpillar DAT cDNA A caterpillar catecholamine transporter cDNA was iden- tified and cloned by RT-PCR using degenerate primers designed to conserved regions of mammalian dopamine and GABA transporters with first-strand cDNA from T. ni caterpillar heads as template. An initial 612 bp amplifica- tion product showed strong similarity to Drosophila and Caenorhabditis elegans DATs in the GenBank database. From this fragment, primers were designed for use in combination with vector primers in nested RACE-PCR to amplify the 5¢ and 3¢ ends of the cDNA. The template for the RACE-PCR was double-stranded head cDNA ligated to plasmid pBK-CMV (Stratagene). The resulting 5¢ RACE-PCR product was approximately 700 bp in length, and the 3¢ RACE-PCR product approximately 3200 bp in length. A complete cDNA sequence of 3.8 kb was assembled by joining the two RACE sequences at a region of overlap in the center of the original 612 bp sequence. Two primers were then designed at the outer ends of the full sequence and used to amplify a fragment of approximately 3500 bp from caterpillar head first-strand cDNA. To ensure the accuracy of the sequence data, this amplification was performed in triplicate and the product cloned and sequenced from each. The final deduced sequence represents the consensus derived by comparing the three independently generated products (GenBank accession #AY154398). 666 P. Gallant et al.(Eur. J. Biochem. 270) Ó FEBS 2003 The cDNA sequence was found to contain an ORF of 1839 bp encoding a potential 612 amino acid protein. The putative translational start site for this ORF was selected based on the presence of an in-frame stop codon located immediately upstream of it. The protein encoded by the ORF (TrnDAT) shows a high degree of similarity to known vertebrate and invertebrate DATs (Fig. 1). Overall identity measured using the ALIGN program [16] to DATs from Drosophila melanogaster (73%), C. elegans (51%), Danio rerio (46%), and human (48%), represent the most closely related sequence for each organism. The greatest variability among the sequences is seen at the termini and in the large second extracellular loop (between TMD3 and TMD4). TrnDAT is also similar to transporters of other monoamines. The amino acid sequence is 49% identical to D. melanogaster SERT, 38% to C. elegans SERT, 47% to Rana catesbeiana ET, 50% to human NET, and 45% identical to T. ni OAT. However, a phylogenetic comparison of the TrnDAT with other invertebrate Na + /Cl – -dependent transporters (Fig. 2) shows that it clusters most closely with other invertebrate DATs. Hydrophobicity analysis of the TrnDAT sequence sug- gests a topology incorporating 12 transmembrane domains (TMDs), with cytoplasmic localization of amino and carboxy termini (not shown). The sequence also possesses many other structural motifs characteristic of Na + /Cl – - coupled biogenic amine transporters, including a heptan leucine zipper motif (L52-L73) within the second TMD [17]. Two conserved cysteine residues C134 and C143 involved in transporter insertion into membranes [18] are present in the second extracellular loop. Conserved residues W37, R38 and C43 present in the first TMD are diagnostic of Na + /Cl – -dependent neurotransmitter transporters and are involved in Na + binding [19]. Also present are conserved residues D32 in the first TMD and S321 and S324 in TMD7, thought to be involved in catecholamine binding [20]. There are two N-glycosylation sites in the second extracellular loop at N155-R158 and N162-S165 of Trn- DAT. Glycosylation has been shown to influence mem- brane trafficking of other transporters [21]. Also present on cytosolic loops of TrnDAT are several putative protein kinase C (S229, T468, T549) and protein kinase A (T15, Fig. 1. Amino acid sequence alignment of the moth dopamine transporter (TrnDAT) with other known DATs. The alignment was performed using CLUSTALX (1.81) and shaded using BOXSHADE (3.21). Identical residues are shaded black when there is a consensus of at least four of the sequences, and similar residues are shaded grey. Bars are drawn over the putative transmembrane domains as predicted for the TrnDAT sequence using TMPRED. The accession numbers of the sequences used are: moth (TrnDAT, AY154398), fruitfly (DrmDAT, AAF76882), nematode (CaeDAT, Q03614), zebrafish (DarDAT, AAK52449), and human (hDAT, AAA19560). Ó FEBS 2003 Monoamine transporters in the moth CNS (Eur. J. Biochem. 270) 667 T562, S590) phosphorylation sites thought to regulate transporter localization [7,22–24]. Characterization of TrnDAT expression Expression of TrnDAT mRNA was assessed by Northern blot analysis (Fig. 3). A single band estimated to be approximately 4.3 kb was detected in head mRNA using a probe representing the TrnDAT cDNA ORF, but was absent in fat body or epidermal mRNA. The size of the band suggests that the RACE-PCR products obtained did not represent the full untranslated regions of the TrnDAT mRNA (4.3 kb vs. 3.8 kb). DIG-labeled TrnDAT cRNA was used as a probe for the cellular localization of transporter mRNA in whole mounts of the caterpillar CNS by in situ hybridization. A fragment of TrnDAT cDNA containing the ORF was used to generate the cRNA through in vitro transcription. A consistent pattern of labeled cell bodies was seen when antisense cRNA was used as the probe, while sense cRNA failed to label cells in the CNS (not shown). A total of approximately 91 cell bodies expressing the TrnDAT transcript were observed in the caterpillar CNS (Figs 4 and 5). The supra-esophageal ganglion (brain) showed the greatest numbers of positive cells (Fig. 4) and they were generally grouped in several paired clusters. The numbers of cells per cluster varied in different brain preparations, and the typical number of cells per cluster is represented in the camera lucida interpretation shown in Fig. 5. The numbers of positive cells observed in the ganglia were smaller than in the brain and were also more consistent from preparation to preparation (Fig. 5, DAT). The camera lucida representa- tion of cell expression of TrnDAT in the caterpillar brain and segmental ganglia is shown in comparison to the previously determined expression pattern for TrnOAT [12] in Fig. 5. Functional comparison of TrnDAT and TrnOAT The functional properties of TrnDAT and TrnOAT were compared using transient expression in recombinant bacu- lovirus-infected Sf9 cells. [ 3 H]DA uptake by cells expressing either transporter was tested at DA concentrations between 0.1 and 20 l M (Fig. 6). Na + -independent (background) accumulation of [ 3 H]DA by TrnDAT- and TrnOAT- expressing cells was assessed in cells incubated in Na + -free saline or in cells mock-infected with virus expressing a-glucuronidase (GUS) protein (Fig. 6). [ 3 H]DA binding Fig. 2. Phylogenetic analysis of neuroactive monoamine transporters. The amino acid sequences of a highly conserved region between TMD4 and TMD8 from a selected group of monoamine transporters were aligned using CLUSTALX (1.81) and an unrooted tree calculated using the neighbor joining method employed by the program. Due to the lack of invertebrate sequences available, some of the sequences selected are not complete cDNAs. However, the region used for comparison was present in every sequence and was chosen for a high level of conservation and minimum of gaps. Confidence values for the derived tree were determined by bootstrapping the dataset using 1000 replicates and a generator seed value of 333 ( CLUSTALX 1.81). The alignment was displayed using TREEVIEW (1.6.5). The accession numbers of the aligned sequences are: moth (TrnDAT, AY154398; TrnOAT, AAL09578), fruitfly (DrmDAT, AAF76882; DrmSERT, AAD10615), mosquito (AngDAT, EAA04277; AngSERT, EAA05837), nematode (CaeDAT, Q03614; CaeSERT, AAK84832), sea hare (ApcSERT, AAK94482), bullfrog (RacET, AAB67676), zebrafish (DarDAT, AAK52449), and human (hDAT, AAA19560; hSERT, AAA35492; hNET, P23975). Fig. 3. Northern blots. T. ni mRNA (8 lg) extracted from caterpillar head, fat body, and epidermis was hybridized with a 32 P-labeled probe representing the ORF of the TrnDAT cDNA. RNA from the head preparation produced a band of approximately 4.3 kb on Kodak Biomax MS film. 668 P. Gallant et al.(Eur. J. Biochem. 270) Ó FEBS 2003 by GUS-virus infected cells was low and failed to saturate at high DA concentrations, similar to that seen in TrnDAT- and TrnOAT-expressing cells exposed to [ 3 H]DA in Na + - free saline. Na + -dependent DA uptake by cells expressing either transporter began to saturate at DA concentrations above 3 l M . The presence of saturable DA uptake was a direct consequence of infection with TrnDAT or TrnOAT recombinant virus. TrnDAT and TrnOAT showed simi- lar and high affinity for DA. The K DA m for TrnDAT was 2.43 ± 0.63 l M (n ¼ 6) over a V max range of 5.1–10.9 nmol DA uptakeÆwell )1 Æmin )1 .TheK DA m for Trn- OAT was 2.16 ± 0.65 l M (n ¼ 4) over a V max range of 5.5–6.3 nmol DA uptakeÆwell )1 Æmin )1 . The cation and anion dependency of DA transport by TrnDAT was similar to published data for TrnOAT [12]. [ 3 H]DA uptake in each replacement saline was normalized to uptake in control saline containing 100 m M Na + and 92.7 m M Cl – . DA uptake by TrnDAT-expressing cells in salines in which equimolar K + ,Li + , choline + or NMG + substituted for Na + dropped to 4.4, 2.0, 1.7 and 1.9%, respectively, of the uptake seen in Na + -containing saline (n ¼ 3). Like TrnOAT, the activity of TrnDAT is abso- lutely Na + dependent. Substitution of Br – or I – for Cl – reduced DA uptake by cells expressing TrnDAT to 34.8% or 15.3% control uptake, respectively. Substitu- ting saline Cl – with PO 4 – ,HCO 3 – ,NO 3 – or gluconate – dropped DA uptake to 7.1, 38.9, 22.0 and 8.2% of control levels, respectively (n ¼ 3). The ability of anions to substitute for Cl – in TrnDAT is ranked as: HCO 3 – >Br – >NO 3 – >I – > gluconate > PO 4 – .DA uptake by TrnOAT-expressing cells can also be supported by saline containing these cation and anion substitutes [12]. In a separate set of experiments, it was found that DA uptake did not saturate at Na + concentrations as high as 153 m M (data not shown), indicating that the K TrnDAT m for Na + must be greater than 100 m M ,asseeninTrnOAT[12]. The uptake of [ 3 H]DA by cells expressing TrnDAT and TrnOAT was inhibited by the five putative competitive transport substrates tested, DA, OA, TA, NE and D -amphetamine (AM) (Fig. 7). The IC 50 values for TrnDAT determined by Hill analysis were 0.6 ± 0.1 l M for AM, 2.3 ± 1.0 l M for DA, 105.8 ± 25.1 l M for OA, 10.0 ± 3.2 l M for TA, and 16.7 ± 6.0 l M for NE. The Fig. 5. Composite camera lucida drawing of cells detected in the cater- pillar CNS by in situ hybridization using TrnDAT cRNA (DAT draw- ing). Cells located dorsally are filled while cells located ventrally are open. OAT-expressing cells (OAT drawing) are shown for comparison and are taken from Malutan et al. [12]. SOG, subesophageal ganglion; T1,first,T2,second,T3,thirdthoracicganglia;A1,first,A2-5,second to fifth abdominal ganglia; TAG, terminal abdominal ganglion. Fig. 4. In situ hybridization of T. ni brain with a TrnDAT antisense RNA. Whole mount preparation of a caterpillar brain was hybridized with a DIG-labeled cRNA representing the TrnDAT ORF and detection was accomplished with an alkaline phosphatase-linked DIG antibody and BCIP/NBT substrate. Positively stained cells were found to be grouped in several loosely defined clusters mirrored between the two lobes. Ó FEBS 2003 Monoamine transporters in the moth CNS (Eur. J. Biochem. 270) 669 corresponding K i values are listed in Table 1. The rank order of inhibitor influence for DA uptake by TrnDAT was AM > DA > TA > NE  OA. The IC 50 values for TrnOAT were 0.5 ± 0.1 l M for AM, 2.4 ± 0.7 l M for DA, 1.8 ± 0.4 l M for OA, 0.5 ± 0.2 l M for TA, and 18.3 ± 1.1 l M for NE. The corresponding K i values are listed in Table 1. The rank order of inhibitor potency on DA uptake by TrnOAT was TA ¼ AM > OA > DA  NE. The ratio of these inhibition data (K DAT i =K OAT i ) indicates that TrnOAT has a 62-fold greater affinity for OA and a 22-fold greater affinity for TA (i.e. a selective affinity for monohydroxy- over dihydroxyphenolamines) than TrnDAT, DrmDAT or CaeDAT (Table 1). Both moth transporters appear to have similar affinities for the other compounds tested (K DAT i =K OAT i  1). Fifteen compounds known to block monoamine uptake in the mammalian CNS were examined for their ability to suppress [ 3 H]DA uptake by cells expressing either moth DAT or OAT. The most potent blockers tested were all found to be TrnDAT-selective (Fig. 8). A complete list of the compounds tested is given in Table 2. Nisoxetine, nomifensine, and several dibenzazepines (desipramine, Fig. 6. Saturation kinetics of TrnDAT and TrnOAT-induced accumu- lation of [ 3 H]DA by Sf9 cells transiently infected with recombinant baculovirus. The curves for Na + -dependent uptake of [ 3 H]DA by TrnDAT and TrnOAT (upper curves) are corrected for uptake by cells expressing TrnDAT or TrnOAT in the absence of Na + (lower curves). This background uptake is similar to nonspecific uptake seen in Sf9 cells infected with baculovirus expressing a-glucuronidase (GUS) instead of transporter transcripts. Na + -dependent DA uptake by both transporters saturates below 10 l M .TheK DA m values for TrnDAT and TrnOAT were 2.43 ± 0.63 l M (mean ± SD of six experiments) and 2.16 ± 0.65 l M (mean ± SD of four experiments), respectively. Table 1. Inhibition of [ 3 H]DA uptake by caterpillar DAT and OAT and other invertebrate DATs by structurally-related phenolamines and cate- cholamines. K i (l M ) K i (l M ) TrnDAT TrnOAT Selectivity a DrmDAT b CaeDAT c OA 94±23 1.5±0.4 62 281 67 DA 2.1±0.9 2.1±0.6 1.0 2.9 0.2 TA 8.9±2.9 0.4±0.1 22 23 – NE 14.8±5.3 16.1±0.9 0.9 49 1.2 AM 0.5±0.1 0.4±0.1 1.3 6.6 3.3 a Selectivity ratio calculated as K DAT i =K OAT i . b Po ¨ rzgen et al. [11]. c Jayanthi et al. [37]. Fig. 7. Phenolamine inhibition of [ 3 H]DA uptake by Sf9 cells expressing TrnDAT (top) or TrnOAT (bottom). Data are shown as a percentage of DA uptake in the absence of competitive inhibitor after correction for Na + -independent DA uptake. The data represent the mean ± SD of three to six separate experiments performed on parallel cultures of infected cells. 670 P. Gallant et al.(Eur. J. Biochem. 270) Ó FEBS 2003 imipramine and clomipramine) are potent blockers of TrnDAT. The concentration of nisoxetine and nomifensine required to inhibit 50% DA uptake by TrnDAT was 60- to 90-fold less than that needed to block 50% DA uptake by TrnOAT (Fig. 8 and Table 2). Imipramine displayed the greatest selectivity (IC OAT 50 =IC DAT 50 ratio, Table 2). GBR12909, a potent blocker of DA uptake by mammalian DATs, is a weak blocker of moth DAT and OAT (Table 2) and of fly DAT [11]. Three phenyltropane derivatives, cocaine, 3a-[(4-chlorophenyl)methoxy]tropane (CPTH), and 4¢,4¢-dibromobenztropine (DBBT) were shown to be weak and relatively nonselective blockers of both moth transporters, although all three had greater affinity for TrnDAT (Table 2). Two benzylamine blockers of Na + - dependent octopamine uptake in the cockroach CNS [25], xylamine (Table 2) and DSP-4 (data not shown) had little effect on DA uptake by cells expressing either TrnDAT or TrnOAT. In addition, four antipyschotics were screened for their ability to block DA uptake at 1 l M concentration. Chlorpromazine and chlorprothixene inhibited greater than 90% DA uptake by DAT whereas thioridazine and trifluoperazine were ineffective. At this concentration, none of these four phenothiozines reduced DA uptake by cells expressing TrnOAT. Discussion This paper provides a comparison of the properties of the T. ni DA transporter cloned here and TrnOAT, a high affinity phenolamine transporter that we previously cloned from the cabbage looper moth [12]. The mRNAs and deduced ORFs for these genes indicate they are distinct proteins that contain many conserved structures diagnostic of Na + /Cl – -dependent neurotransmitter transporters. Phy- logenetic analysis of known amine transporters (Fig. 2) clearly distinguishes the transporters of the indolamine 5-HT, representing a functional class that seems to be present in all metazoan organisms examined. Invertebrate transporters of dopamine are also well resolved, and TrnDAT groups quite closely with other insect examples. However, in the resulting tree vertebrate DATs group most closely with transporters of epinephrine and norepineph- rine, compounds that are little used in the invertebrate nervous system. The occurrence of an OAT in the cabbage looper suggests DATs may also have had an alternative route of diversification within the invertebrate lineage. OAT is positioned intermediate between the invertebrate DAT group and the vertebrate DATs and NETs in the phylogeny in Fig. 2. Comparisons with complete sequences show some of the highest levels of identity for OAT are with vertebrate NETs (up to 50% identity, as compared to only 45% identity with TrnDAT). OA in invertebrates plays in many ways a similar role to NE/E in vertebrates, suggesting OAT in the cabbage looper may represent the invertebrate equivalent of vertebrate NETs. Fig. 8. Differential sensitivity of TrnDAT and TrnOAT to selective blockers of high-affinity catecholamine re-uptake in the mammalian CNS. The mammalian NET-selective blockers nisoxetine, desipramine and imipramine and the DAT-selective blocker nomifensine were the most potent blockers tested on TrnDAT. They were about 100-fold less potent in blocking DA uptake by TrnOAT (see Table 2 for details). Data are expressed as percentage Na + -dependent uptake and are the mean ± SD of three to five separate experiments. Table 2. Selectivity of drugs blocking [ 3 H]DA uptake by DAT and OAT. Chemical structure Compound IC DAT 50 (n M ± SD) IC OAT 50 (n M ± SD) Selectivity index a Phenoxypropanamine Nisoxetine 9 ± 4 800 ± 290 89 Fluoxetine 540 ± 170 7000 ± 1,100 13 Phenylisoquinolinamine Nomifensine 26 ± 11 1600 ± 100 62 Dibenzazepine b Desipramine 45 ± 8 2000 ± 500 44 Imipramine 48 ± 6 8300 ± 3,900 173 Clomipramine 58 ± 10 5600 ± 2,300 96 Maprotiline 830 ± 200 16 000 ± 2,200 19 Naphthyridine carboxylate Amfonelate 380 ± 50 8200 ± 700 22 Diphenylmethyl oxyalkylpiperazine GBR12909 3,100 ± 400 8100 ± 500 3 Cyclohexylpiperidine BTCP 610 ± 230 18 000 ± 3,000 29 Phenyltropane Cocaine 7,000 ± 550 70 000± 24,000 10 CPTH 7,400 ± 440 40 000 >5 DBBT 9,500 ± 4,400 18 000 ± 3,500 2 Benzylamine Bupropion 15,000 ± 4,000 >100 000 >6 Xylamine >200,000  40 000 1 a Selectivity index based on IC 50 values (IC OAT 50 =IC DAT 50 ). b ÔTricyclic antidepressantsÕ. Ó FEBS 2003 Monoamine transporters in the moth CNS (Eur. J. Biochem. 270) 671 TrnDAT is expressed primarily in the CNS of T. ni.In insects, dopaminergic neurons have been identified immu- nocytochemically in D. melanogaster [26,27], Gryllus bima- culatus [28], Apis mellifera [29], and Schistocerca gregaria [30]. The existence of dopaminergic neurons in the insect CNS implies that a dopamine transporter would need to be expressed by these neurons to clear the chemical from the synaptic space. Octopamine is also an accepted neurotrans- mitter in the insect CNS [1], and octopaminergic neurons [31] and several octopamine receptors [2,32] have been identified in the fly and other insects. At the cellular level, TrnDAT and TrnOAT RNAs are expressed by different sets of neurons in the caterpillar CNS. This suggests that moth DAT and OAT are functionally distinct and are expressed by neurons constituting different aminergic pathways in the moth CNS. The brain and each segmental ganglion of the looper caterpillar CNS contained cells with TrnDAT transcripts. The number of DAT-positive cell bodies in the caterpillar brain is similar to that in adult fly and locust [30,33]. Fewer cell clusters are seen in the brain in the fly maggot. By comparison, TrnOAT is expressed by few cell bodies in the caterpillar brain (Fig. 5 [12]). TrnOAT RNA is expressed in octopaminergic neurons, as shown by its colocalization with transcripts for tyramine b-hydroxylase, a marker enzyme of OA-signaling pathways [12]. Cell bodies expressing Trn- OAT are most numerous in the subesophageal ganglion. Each of the thoracic and abdominal ganglia in the ventral nerve cord of larval T. ni contain cell bodies expressing TrnDAT transcripts. TrnDAT is most strongly expressed in the ventral nerve cord in ganglion T1. This ganglion contains the largest number of dopaminergic cell bodies in the ventral nerve cord of adult Drosophila [33]. TrnOAT differs markedly from TrnDAT in its lack of expression in the abdominal ganglia other than ganglion A1. It remains to be shown what system for OA uptake functions in the abdominal nerve cord of the caterpillar. The absence of an OAT in the fly genome implies that some other mechanism of OA clearance must be available. Po ¨ rzgen et al.[11] suggestthatinDrosophila extracellular OA may be taken up by low-affinity cation transporters or degraded by enzymes. Clearly, such a system will be unrelated to the known Na + /Cl – -dependent transporter archetype, and provides no clues as to why the moth has need for independent high affinity-type DATs and OATs. Neurotransmitter transporters are normally named after their tissue context and/or substrate they transport most efficiently [12,34]. Although TrnDAT and TrnOAT have similar affinities for DA (K m ¼ 2.43 and 2.16 l M , respect- ively, within the range reported for cloned mammalian DATs), our data suggest that DA is the primary natural substrate of TrnDAT and octopamine (and possibly tyramine) the natural substrate of TrnOAT (K OA m ¼ 2.05 l M [12]). DA uptake by TrnOAT is 62 times more sensitive to OA than is DA uptake by TrnDAT. Thus, while TrnOAT might in principle use DA as a high-affinity transport substrate were this transporter expressed at appropriate sites in the CNS, TrnDAT is unlikely to play a reciprocal role by doubling up as an OA transporter in situ. The role of TA, a precursor of OA in octopaminergic neurons, as a neurotransmitter in the insect nervous system is less clear [1]. Both moth DAT and OAT are relatively insensitive to cocaine. In mammals, submicromolar concentrations of cocaine inhibit the Na + -dependent uptake of NE and DA by their respective cloned transporters [34–36]. DA transporters cloned from other invertebrates are also less sensitive to cocaine. The cocaine concentration required to reduce the uptake of DA by moth DAT by 50% (IC 50 ¼ 7.0 l M ) is similar to that reported for fly DAT (IC 50 ¼ 2.7 l M [11]) and worm DAT (IC 50 approximately 5 l M [37]), about one order of magnitude greater than that reported to block DA uptake by human DAT, or NE uptake by hNET. Moth OAT is even more resistant to cocaine inhibition (IC 50 ¼ 70 l M ). These findings fail to support the notion that the neuronal octopamine uptake system is the main insecticidal target of cocaine [13,38]. This notion was based on the finding that the Na + -dependent uptake of DA by synaptosomes isolated from the nervous system of the cockroach Blaberus waslesssensitiveto cocaine inhibition (IC 50 )100 l M ) than synaptosomal Na + - dependent OA uptake (IC 50 )40 l M ) [38]. Cocaine, how- ever, disrupts serotonin transport in the fly at nanomolar levels (K i ¼ 0.5 l M [9,11]). Xylamine, reported to block Na + -dependent OA uptake in the cockroach CNS [25] is also an ineffective blocker of DA uptake by TrnDAT. As noted by Po ¨ rzgen et al. [11], the invertebrate dopamine transporters DrmDAT and CaeDAT have pharmacological features intermediate between those of mammalian DATs, NETs and SERTs. Nisoxetine is a potent blocker of dopamine uptake by invertebrate DATs, but a relatively weak blocker of dopamine uptake by mammalian DATs. The tricyclic antidepressants desipr- amine and imipramine are potent blockers of DA uptake by invertebrate DATs [11,37]. In mammals they are strong and selective blockers of NET [35,39,40] and weak blockers of DAT [36,39–41]. GBR12909, a potent blocker of dopamine uptake by mammalian DATs is a weak blocker of TrnDAT and other invertebrate DATs [11]. Cocaine is a powerful blocker of hDAT but apparently not of inver- tebrate DATs. Nomifensine, on the other hand, is a selective blocker of both mammalian DATs [42,43] and invertebrate DATs (moth (present data) and nematode [37]). TrnDAT has a pharmacological profile similar to that of other invertebrate DATs but distinct from that of mammalian DATs. The enigmatic pharmacological profile of TrnOAT does not allow it to fit easily into this model. It is at best only weakly sensitive to drugs that selectively inhibit the high affinity uptake of monoamine neurotrans- mitters by members of the Na + /Cl – -dependent transporter family cloned from other organisms. The sequence analysis of TrnOAT suggests that it lies on a separate branch in this transporter family and potential homologs are apparently absent from the genomes of flies and nematodes. While it is possible that the DrmDAT gene is a descendant of an ancestral invertebrate gene that subsequently duplicated to give rise to both classes of vertebrate catecholamine transporter genes [11], our data show that the genomes of insects such as the cabbage looper possess at least two distinct genes that code for high-affinity transporters of neuronal catecholamines and phenolamines. Furthermore, pharmacological studies suggest that OA and DA are transported by different Na + -dependent mechanisms in the CNS in insects such as the cockroach [13,25,38]. The 672 P. Gallant et al.(Eur. J. Biochem. 270) Ó FEBS 2003 TrnOAT gene might represent an ancient gene that encoded a nonselective phenolamine/catecholamine trans- porter, or alternatively, it could have derived subsequently from an ancestral catecholamine-selective transporter more closely related to invertebrate DATs. 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