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:
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. 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.
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