Functionally distinct dopamine and octopamine transportersin the CNS of the cabbage looper moth* Pamela Gallant1,2, Tabita Malutan1,2, Heather McLean2, LouAnn Verellen1, Stanley Caveney2
Trang 1Functionally distinct dopamine and octopamine transporters
in the CNS of the cabbage looper moth*
Pamela Gallant1,2, Tabita Malutan1,2, Heather McLean2, LouAnn Verellen1, Stanley Caveney2
and Cam Donly1
1
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, and2Department 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 3.8kb
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 elegansDAT (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 (Km
2.43 and 2.16 lM, respectively) However, the competitive
substrates octopamine and tyramine are more potent
blockers of [3H]dopamine (DA) uptake by TrnOAT than by
TrnDAT.D-Amphetamine is a strong inhibitor andL
-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)
Trang 2one 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 mM dNTPs, 2.5 mM MgCl2,
2 pmolÆmL)1 degenerate primers, 2.5 U Taq DNA
Poly-merase (Life Technologies, Burlington, ON, Canada) The
PCR conditions were 94C for 2 min, followed by 35 cycles
of 94C for 45 s, 55 C for 45 s and 72 C for 1 min,
followed by one 5-min hold at 72C RACE-PCR
contained 350 lM dNTPs, 0.4 pmolÆmL)1 primers,
0.75 mM MgCl2, 1X Expand buffer 3 (contains 2.25 mM
MgCl2), and 2.5 U Expand enzyme (Expand Long
Tem-plate PCR System, Roche Diagnostics, Laval, QC,
Canada) Cycling conditions were 94C for 2 min, followed
by 10 cycles of 94C for 20 s, 62–65 C for 30 s, and 68 C
for 3 min, followed by 12 cycles of 94C for 20 s, 62–65 C
for 30 s, and 68C for 3 min + 20 s increment per cycle,
followed by a 20-min hold at 68C 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 lL of 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 32P 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· 106d.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 C to Kodak BioMax MS film for 18h
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 NcoI or NdeI (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/Pi(130 mMNaCl,
70 mMNa2HPO4, 3 mMNaH2PO4, pH 7.4) Tissues were fixed in 4% paraformaldehyde in NaCl/Pifor 2–3 h and stored at 4C in NaCl/Pi/Triton (0.3% Triton X-100 in NaCl/Pi) until use In situ hybridizations were performed essentially as described by Malutan et al [12] using
175 ngÆmL)1of 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
Trang 3Technologies) 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 27C for 48h postinfection The cells were
washed for 1 h with Na+-containing saline (11.2 mMMgCl2,
11.2 mMMgSO4, 53.5 mMNaCl, 7.3 mMNaH2PO4, 55 mM
KCl, 76.8mM sucrose, pH to 7.0 with KOH) and then
incubated for 3 min in 500 lL saline to which [3H]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 lL of 1 mCiÆmL)1[3H]DA added to saline on ice
to give a final concentration of 0.3 lM[3H]DA (except for the
two lowest concentrations used in the kinetic analysis)
Solutions were warmed to 27C immediately before use DA
uptake was terminated by washing the cells several times in
Na+-free saline (11.2 mM MgCl2, 11.2 mM MgSO4,
53.5 mMcholine chloride, 7.3 mMKH2PO4, 55 mM KCl,
76.8mMsucrose, 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 [3H]DA accumulation at 12 DA concentrations from 0.1
to 20 lMDA Cells exposed to 0.1, 0.2 and 0.3 lMDA were
incubated in saline containing [3H]DA only (1.67, 3.33 and
5 lL of 1 mCiÆmL)1[3H]DA) [3H]DA was supplemented
with unlabeled DA to give the higher DA concentrations
The affinities of the two transporters for DA (KDA
m ) and maximum rates of DA uptake (Vmax) by virally infected cells
were estimated through Eadie–Hofstee transformation of the
[3H]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 [3H]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–, NO3, PO4 or HCO3 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 [3H]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% (IC50
value) was determined by Hill analysis (where inhibitor
concentration is plotted against I/Imax – I on a double
logarithmic plot, with I¼ inhibition and Imax¼ maximum
inhibition) The TrnDAT and TrnOAT inhibition constants
(K) for each competitive inhibitor were derived from the IC
values using the Cheng and Prusoff [15] equation,
IC50¼ Kið1 þ ½S=kDA
m Þ, where [S] is the [3H]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 [3H]DA uptake by cells expressing TrnDAT or TrnOAT The cells were exposed to inhibitor alone for 4 min before incubation with [3H]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 [3H]DA accumulation was normalized to DA uptake by untreated cells after correcting for the radioactivity associated with cells exposed
to [3H]DA in Na+-free saline IC50 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), clomiprN-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine 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 3.8kb 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)
Trang 4The 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 melanogasterSERT, 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-R158and 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).
Trang 5T562, 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 3.8kb)
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 [3H]DA uptake by cells expressing either transporter was tested at DA concentrations between 0.1 and 20 lM (Fig 6) Na+-independent (background) accumulation of [3H]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) [3H]DA binding
Fig 2 Phylogenetic analysis of neuroactive monoamine transporters.
The amino acid sequences of a highly conserved region between
TMD4 and TMD8from 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.
Trang 6by 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 [3H]DA in Na+
-free saline Na+-dependent DA uptake by cells expressing
either transporter began to saturate at DA concentrations
above 3 lM 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 KDA
was 2.43 ± 0.63 lM (n¼ 6) over a Vmax range of
5.1–10.9 nmol DA uptakeÆwell)1Æmin)1 The KDA
m for Trn-OAT was 2.16 ± 0.65 lM (n¼ 4) over a Vmaxrange of
5.5–6.3 nmol DA uptakeÆwell)1Æmin)1
The cation and anion dependency of DA transport by
TrnDAT was similar to published data for TrnOAT [12]
[3H]DA uptake in each replacement saline was normalized
to uptake in control saline containing 100 mM Na+and
92.7 mM 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
(n¼ 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 PO4, HCO3, NO3 or gluconate–
dropped DA uptake to 7.1, 38.9, 22.0 and 8.2% of
control levels, respectively (n¼ 3) The ability of anions
to substitute for Cl– in TrnDAT is ranked as:
HCO3 > Br–> NO3 > I–> gluconate > PO4 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 mM(data not shown), indicating that the KTrnDAT
Na+must be greater than 100 m , as seen in TrnOAT [12]
The uptake of [3H]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 IC50 values for TrnDAT determined by Hill analysis were 0.6 ± 0.1 lM for AM, 2.3 ± 1.0 lMfor DA, 105.8± 25.1 lMfor OA, 10.0 ± 3.2 l for TA, and 16.7 ± 6.0 l 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, third thoracic ganglia; 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.
Trang 7corresponding Ki values are listed in Table 1 The rank
order of inhibitor influence for DA uptake by TrnDAT
was AM > DA > TA > NE OA The IC50values for
TrnOAT were 0.5 ± 0.1 lM for AM, 2.4 ± 0.7 lM for
DA, 1.8± 0.4 lM for OA, 0.5 ± 0.2 lM for TA, and
18.3 ± 1.1 lM for NE The corresponding Ki values are
listed in Table 1 The rank order of inhibitor potency on
DA uptake by TrnOAT was TA¼ AM > OA > DA
NE The ratio of these inhibition data (KDAT
i =KOAT
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 (KDAT
i =KiOAT 1)
Fifteen compounds known to block monoamine uptake
in the mammalian CNS were examined for their ability to suppress [3H]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 [3H]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 The K 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.
TrnDAT TrnOAT Selectivity a DrmDAT b CaeDAT c
a Selectivity ratio calculated as K DAT =K OAT b Po¨rzgen et al [11] c Jayanthi et al [37].
Fig 7 Phenolamine inhibition of [3H]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.
Trang 8imipramine 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 8and Table 2) Imipramine displayed the
greatest selectivity (ICOAT50 =ICDAT50 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 lM 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 [3H]DA uptake by DAT and OAT.
Chemical structure Compound ICDAT50 (n M ± SD) ICOAT50 (n M ± SD) Selectivity indexa Phenoxypropanamine Nisoxetine 9 ± 4 800 ± 290 89
Fluoxetine 540 ± 170 7000 ± 1,100 13 Phenylisoquinolinamine Nomifensine 26 ± 11 1600 ± 100 62
Dibenzazepineb Desipramine 45 ± 82000 ± 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 18000 ± 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 18000 ± 3,500 2 Benzylamine Bupropion 15,000 ± 4,000 >100 000 >6
Xylamine >200,000 40 000 1
a
Selectivity index based on IC values (ICOAT=ICDAT).bTricyclic antidepressants.
Trang 9TrnDAT 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]
suggest that in Drosophila 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 (Km¼ 2.43 and 2.16 lM,
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 (KOA
2.05 lM [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% (IC50¼ 7.0 lM) is similar to that reported for fly DAT (IC50¼ 2.7 lM[11]) and worm DAT (IC50approximately
5 lM[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 (IC50¼ 70 lM) 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 was less sensitive to cocaine inhibition (IC50)100 lM) than synaptosomal Na+ -dependent OA uptake (IC50)40 lM) [38] Cocaine, how-ever, disrupts serotonin transport in the fly at nanomolar levels (Ki¼ 0.5 lM [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 imiprdesipr-amine 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
Trang 10TrnOAT 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 Further work on the
molecular biology of monoamine transporters expressed in
the CNS of basal metazoan orders is needed before
predictions as to the nature of the primordial
catecholam-ine transporter gene(s) can be made with any certainty
Acknowledgments
These studies were supported by Agriculture and Agri-Food Canada
(C D.), by the Natural Sciences and Engineering Research Council of
Canada (S C.) and Aventis CropScience.
References
1 Osborne, R.H (1996) Insect neurotransmission:
Neurotransmit-ters and their receptors Pharmacol Ther 69, 117–142.
2 Roeder, T (1999) Octopamine in invertebrates Progr Neurobiol.
59, 533–561.
3 Claassen, D.E & Kammer, A.E (1986) Effects of octopamine,
dopamine, and serotonin on production of flight motor output by
thoracic ganglia of Manduca sexta J Neurobiol 17, 1–14.
4 Stevenson, P.A., Hofmann, H.A., Schoch, K & Schildberger, K.
(2000) The fight and flight responses of crickets depleted of
bio-genic amines J Neurobiol 43, 107–120.
5 Sneddon, L.U., Taylor, A.C., Huntingford, F.A & Watson, D.G.
(2000) Agonistic behaviour and biogenic amines in shore crabs
Carcinus maenas J Exp Biol 203, 537–545.
6 Mercer, A.R & Menzel, R (1982) The effects of biogenic amines
on conditioned and unconditioned responses to olfactory stimuli
in the honeybee Apis mellifera J Comp Physiol 145A, 363–368.
7 Masson, J., Sagne´, C., Hamon, M & El Mestikawy, S (1999)
Neurotransmitter transporters in the central nervous system.
Pharmacol Rev 51, 439–464.
8 Caveney S & Donly, B.C (2002) Neurotransmitter transporters
in the insect nervous system Adv Insect Physiol 29, 55–149.
9 Corey, J.L., Quick, M.W., Davidson, N., Lester, H.A &
Guastella, J (1994) A cocaine-sensitive Drosophila serotonin
transporter: cloning, expression, and electrophysiological
char-acterization Proc Natl Acad Sci USA 91, 1188–1192.
10 Demchyshyn, L.L., Pristupa, Z.B., Sugamori, K.S., Barker, E.L.,
Blakely, R.D., Wolfgang, W.J., Forte, M.A & Niznik, H.B.
(1994) Cloning, expression, and localization of a
chloride-facilitated, cocaine-sensitive serotonin transporter from
Droso-phila melanogaster Proc Natl Acad Sci USA 91, 5158–5162.
11 Po¨rzgen, P., Park, S.K., Hirsch, J., Sonders, M.S & Amara, S.G.
(2001) The antidepressant-sensitive dopamine transporter in
Drosophila melanogaster: a primordial carrier for catecholamines.
Mol Pharmacol 59, 83–95.
12 Malutan, T., McLean, H., Caveney, S & Donly, C (2002) A
high-affinity octopamine transporter cloned from the central nervous
system of cabbage looper Trichoplusia ni Insect Biochem Mol.
Biol 32, 343–357.
13 Nathanson, J.A., Hunnicutt, E.J., Kantham, L & Scavone, C.
(1993) Cocaine as a naturally occurring insecticide Proc Natl
Acad Sci USA 90, 9645–9648.
14 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular
Cloning: A Laboratory Manual Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York.
15 Cheng, Y.-C & Prusoff, W.H (1973) Relationship between the
inhibition constant (K I ) and the concentration of inhibitor which
causes 50 per cent inhibition (I 50 ) of an enzymatic reaction Biochem Pharmacol 22, 3099–3108.
16 Pearson, W.R., Wood, T., Zhang, Z & Miller, W (1997) Com-parison of DNA sequences with protein sequences Genomics
46, 24–36.
17 Quick, M.W., Corey, J.L., Davidson, N & Lester, H.A (1997) Second messengers, trafficking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1 J Neurosci 17, 2967–2979.
18 Wang, J.B., Moriwaki, A & Uhl, G.R (1995) Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression J Neurochem 64, 1416–1419.
19 Mager, S., Kleinberger-Doron, N., Keshet, G.I., Davidson, N., Kanner, B.I & Lester, H.A (1996) Ion binding and permeation at the GABA transporter GAT1 J Neurosci 16, 5405–5414.
20 Kitayama, S., Shimada, S., Yu, H., Markham, L.K., Donovan, D.M & Uhl, G.R (1992) Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding Proc Natl Acad Sci USA 89, 7782–7785.
21 Bennett, E.R & Kanner, B.I (1997) The membrane topology of GAT-1, a (Na + + Cl – )-coupled c-aminobutyric acid transporter from rat brain J Biol Chem 272, 1203–1210.
22 Beckman, M.L & Quick, M.W (1998) Neurotransmitter trans-porters: regulators of function and functional regulation.
J Membr Biol 164, 1–10.
23 Liu, Y., Krantz, D.E., Waites, C & Edwards, R.H (1999) Membrane trafficking of neurotransmitter transporters in the regulation of synaptic transmission Trends Cell Biol 9, 356–363.
24 Blakely, R.D & Bauman, A.L (2000) Biogenic amine transpor-ters: regulation in flux Curr Opin Neurobiol 10, 328–336.
25 Wierenga, J.M & Hollingworth, R.M (1990) Octopamine uptake and metabolism in the insect nervous system J Neurochem 54, 479–489.
26 Lundell, M.J & Hirsch, J (1994) Temporal and spatial develop-ment of serotonin and dopamine neurons in the Drosophila CNS Dev Biol 165, 385–396.
27 Monastirioti, M (1999) Biogenic amine systems in the fruit fly Drosophila melanogaster Microsc Res Techn 45, 106–121.
28 Ho¨rner, M., Sporhase-Eichmann, U., Helle, J., Venus, B & Schurmann, F (1995) The distribution of neurons immuno-reactive for b-tyrosine hydroxylase, dopamine and serotonin in the ventral nerve cord of the cricket, Gryllus bimaculatus Cell Tissue Res 280, 583–604.
29 Scha¨fer, S & Rehder, V (1989) Dopamine-like immunoreactivity
in the brain and suboesophageal ganglion of the honeybee.
J Comp Neurol 280, 43–58.
30 Wendt, B & Homberg, U (1992) Immunocytochemistry of dopamine in the brain of the locust Schistocerca gregaria J Comp Neurol 321, 387–403.
31 Monastirioti, M., Gorczyca, M., Rapus, J., Eckert, M., White, K.
& Budnik, V (1995) Octopamine immunoreactivity in the fruit fly Drosophila melanogaster J Comp Neurol 356, 275–287.
32 Arakawa, S., Gocayne, J.D., McCombie, W.R., Urquhart, D.A., Hall, L.M., Fraser, C.M & Venter, J.C (1990) Cloning, locali-zation, and permanent expression of a Drosophila octopamine receptor Neuron 2, 343–354.
33 Budnik, V & White, K (1988) Catecholamine containing neurons
in Drosophila: distribution and development J Comp Neurol.
268, 400–413.
34 Apparsundaram, S., Moore, K.R., Malone, M.D., Hartzell, H.C.
& Blakely, R (1997) Molecular cloning and characterization of an
L -epinephrine transporter from sympathetic ganglia of the bullfrog Rana catesbiana J Neurosci 17, 2691–2702.