Amine Transporters
Juan Zhen, lllinois State University, Normal, lllinois, USA
Maarten EA Reith, New York University School of Medicine, New York, USA
Amine transporters in plasma membranes mediate the transport of dopamine,
(nor)adrenaline ((nor)epinephrine) and serotonin. These transporters are members of the
larger Na+- and Cl2-dependent neurotransmitter transporters.
Introduction
Amine transporters in plasma membranes mediate the
transport of dopamine, nor(adrenaline) ((nor)epinephrine)
and serotonin. They are part of the larger family of Na+-
and Cl2-dependent plasma membrane transporters, which
also includes the transporters for proline, glycine, g-ami-
nobutyric acid (GABA), taurine, betaine, creatine and
choline. Other known amine transporters reside in mem-
branes of storage vesicles, where they mediate the vesicular
uptake of dopamine, noradrenaline or serotonin (Figure 1).
However, the plasma membrane and vesicular amine
Figure 1 Amine transporters in plasma membranes and in membranes of
storage vesicles. The amine transporter in the plasma membrane transports
amine (A) from the extracellular space into the cytoplasm, with cotransport
of Na+ and Cl2. This uptake is driven by the inwardly directed Na+ gradient
and outwardly directed K+ gradient, maintained by Na+/K+ adenosine
triphosphatase (ATPase). Uptake of dopamine and noradrenaline, but not
of serotonin, is also promoted by the membrane potential, negative inside.
In contrast, the vesicular amine transporter (VAT) takes up A with
countertransport of H+, and the high intravesicular H+ concentration is
maintained by the ATP-dependent H+ pump or H+-transporting ATPase (HT
ATPase). On the postsynaptic side, an amine receptor is depicted coupled
to G protein subunits.
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Article Contents
. Introduction
. Transporters for Dopamine, Noradrenaline and
Serotonin
. Amine Transporters: Membrane Proteins with 12
Transmembrane Segments
. Ionic Dependence and Electrogenic Properties of
Amine Transporters
. Regulation of Amine Transporters
. Involvement of Amine Transporters in Psychiatric
Disorders
. MPTP and Parkinson Disease
. Transporters as Targets for Drug Action
. Summary
doi: 10.1038/npg.els.0004091
transporters are entirely different proteins with divergent
function, pharmacology and structure. This paper ad-
dresses the amine transporters in plasma membranes. See
also: Neurotransmitter transporters
Na+- and Cl2-dependent plasma membrane transport-
ers enable the transport of substrate together with cosu-
bstrates Na+ and Cl2 (Figure 1). A transmembrane Na+
concentration gradient is maintained by Na+/K+ adeno-
sine triphosphatase (ATPase) and drives the uptake of
substrate towards the inside of the cytosol where the Na+
concentration is low. In comparison, vesicular amine up-
take is driven by the proton electrochemical gradient gen-
erated by an adenosine triphosphate (ATP)-dependent H+
pump; in the translocation process, protons are counter-
transported with the uptake of amine into the storage ves-
icle (Figure 1). See also: ATPases: ion-motive; Ion motive
ATPases: V- and P-type ATPases
Transporters for Dopamine,
Noradrenaline and Serotonin
Initial indications for uptake of monoamines into nerve
terminals came from early work four decades ago in 1959,
which showed that injected radiolabelled catecholamines
accumulated in peripheral tissues depending on intact
sympathetic terminals. When the blood–brain barrier was
bypassed by injection into lateral ventricles, radiolabelled
catecholamines were found in catecholamine-rich brain
areas. Brain-slice preparations allowed the characteriza-
tion of high-affinity uptake of dopamine, noradrenaline
and serotonin by both kinetic and pharmacological
approaches. See also: Adrenaline and noradrenaline;
Dopamine; Serotonin
1
 Amine Transporters

Early kinetic studies reported on initial velocity deter- Two techniques, developed and refined in recent years,
minations of radiolabelled substrate, showing the satura- have made it possible to study the function of transporters
bility of uptake as a function of substrate concentration in vivo: microdialysis and voltammetry. Both microdialysis
according to Michaelis–Menten kinetics. The affinity of and voltammetry have shown substantial increases in ex-
the neuronal uptake process, represented by the Michaelis tracellular dopamine, noradrenaline and serotonin levels
constant (Km), was in the low micromolar range for all when transporters are blocked by drugs targeting them.
three monoamines, and the uptake of substrate resulted in These results support the concept that monoamine trans-
intracellular concentrations that were many times higher porters play a crucial role in the clearance of released amine
than the applied outside concentrations. Pharmacological transmitter, as borne out by the extremely elongated clear-
characterization in these slice studies indicated distinct ance interval reported for released dopamine in knockout
properties of uptake of each of the three monoamines, mice lacking the DAT (Giros et al., 1996). See also:
suggesting separate uptake systems. See also: Enzyme Knockout and knock-in animals
kinetics: steady state
The next major step in characterizing monoamine up-
take came with the development of preparations rich in
nerve terminals, the synaptosomes, which were shown to
Amine Transporters: Membrane
accumulate radiolabelled monoamines with a Km in the Proteins with 12 Transmembrane
submicromolar range. Early on, it was found that the nor-
adrenaline uptake system, for example, in the cerebral
Segments
cortex and hypothalamus, also mediates the transport of
The coding sequence for the amine transporters covers
dopamine with high affinity, even higher than that of nor-
approximately 2 kb on their respective complementary de-
adrenaline itself (Snyder and Coyle, 1969). The opposite
oxyribonucleic acids (cDNAs), corresponding to 617–630
was not found to be true; preparations of striatal synapto-
amino acids and giving molecular weights of 60–97 kDa
somes, in which dopamine terminals are predominant,
depending on the extent of glycosylation. The primary se-
transport dopamine with substantially higher affinity than
quences of NAT and DAT display a homology of 80%,
noradrenaline. Little overlap was found between the cat-
and those of DAT and SERT 69%, allowing for sequence
echolamine uptake systems for dopamine and noradren-
differences that do not alter the translated amino acids.
aline, on the one hand, and the indoleamine uptake system
The respective sequence identities are 67 and 49%. The AT
for serotonin, on the other. See also: Synaptic vesicles:
displays 75% identity with NAT, 66% with DAT and 48%
methods for preparation
with SERT. Hydropathy analysis of the sequences suggests
Much information has also been derived from plasma
the presence of 12 transmembrane domains throughout the
membrane vesicle preparations, allowing the study of trans-
entire Na+- and Cl2-dependent plasma membrane trans-
port driven by experimenter-manipulated ion gradients
porters (Figure 2). The amine transporters also share with
only in the absence of storage vesicles. Subsequent progress
the other members of this family the intracellular location
in the monoamine uptake area was made when it became
possible to measure the interaction between transporters
and uptake blockers by the application of receptor-binding
techniques (Langer et al., 1980). High-affinity radioligands
were developed for each amine transporter, allowing meas-
urements at the level of recognition. See also: Receptor
binding in drug discovery
The most recent era in monoamine research was initiated
by the cloning of the amine transporters. The cloning of a
related GABA transporter, GAT-1, and the development
of the new expression-cloning technique, made it possible
to clone the noradrenaline transporter (NAT) (Pacholczyk
et al., 1991), which was followed rapidly by the cloning of
the dopamine transporter (DAT), the serotonin transport-
er (SERT) and adrenaline transporter (AT). Expression of
each transporter has now been studied in a variety of cell
lines, after both transient and stable expression, and struc-
ture–activity relationships have been deduced from site- Figure 2 Proposed topology of plasma membrane amine transporters.
directed mutagenesis and chimaera studies (see the section The large extracellular loop between transmembrane domains 3 and 4
have two to four consensus sites for glycosylation (c), whereas consensus
Transporters as targets for drug action). See also: Gene sites for phosphorylation by PKA (A), PKC (C) or Ca2+ –calmodulin-
transfer and Expression in tissue culture cells; Mutagen- dependent kinase II (KII) are found on the amino and carboxy tails as well as
esis: site-specific on the second intracellular loop.

2

of the N- and C-termini and a large glycosylated extracel-
lular loop between transmembrane domains 3 and 4. In
comparison, the vesicular amine transporters, while also
having 12 transmembrane domains, display a large glyco-
sylated hydrophilic loop between transmembrane domains
1 and 2, and show no significant homology with the plasma
membrane amine transporters. See also: Hydrophobicity
plots; Membrane proteins
Recent evidence indicates that the DAT and SERT are
organized in multimeric structures (Torres et al., 2003;
Kilic and Rudnick, 2000; Sitte and Freissmuth, 2003).
Transmembrane domains 2, 6 and 11/12 form likely con-
tact points, with transmembrane 2 contacts involving
a leucine zipper-like motif and transmembrane 6 contacts
being of the homophilic type involving the glycophorin
A motif.
Although multiple messenger ribonucleic acid
(mRNA) species have been described for NAT and
SERT, there appears to be only one gene for each amine
transporter, mapped to chromosome 16q12–q21 for the
NAT, 5p15.3 for the DAT and 17q11.1–q12 for the
SERT. In these genes, most of the transmembrane do-
mains are encoded on a distinct exon as in the GABA
transporter gene. For each monoamine, there is only one
type of transporter in the plasma membrane, unlike the
situation for neurotransmitter receptors that come in
multiple subtypes. However, variants of the amine trans-
porters have recently been described, some of which have
functional implications (see Hahn and Blakely, 2002).
See also: Genome mapping
Ionic Dependence and Electrogenic
Properties of Amine Transporters
Uptake of amine substrate by its plasma membrane trans-
porter is thought to occur in several steps, as depicted in
Figure 3. The transporter in the external-facing form binds
Na+, Cl2 and substrate, allowing a conformational change
in the internal-facing form. Na+, Cl2 and substrate disso-
ciate into the cytoplasm, and the carrier reorients itself. The
latter step is accelerated by K+ or H+ in the case of the
SERT, but probably not for the DAT or NAT. Evidence
has been advanced for all three monoamines that they are
translocated in the cationic protonated form, which is the
predominant form at physiological pH (Berfield et al.,
1999). Most probably, one Na+ and one Cl2 ion are co-
transported with noradrenaline as well as serotonin; in
contrast, two Na+ ions accompany the translocation of
dopamine. See also: Protein translocation across mem-
branes; Transition states: substrate-induced conforma-
tional transitions
Taken together with the likelihood that the cationic form
of substrate is transported and that K+ is countertrans-
ported only for the SERT, it can be deduced that the
Amine Transporters
Figure 3 Translocation cycle. Starting at the lower left and continuing
counterclockwise, the transporter facing outward binds Na+, Cl2 and
amine (A). A conformational change occurs bringing the transporter in a
form facing inward (top right-hand corner) allowing the dissociation of
Na+, Cl2 and A. The empty, inward-facing, transporter undergoes the
reorientation step (top left to bottom left), which in the case of the
serotonin transporter is promoted by K+. The transporter is now ready for a
new translocation cycle.
translocation of dopamine and noradrenaline is elect-
rogenic (bringing in positive charge), whereas that of
serotonin is electroneutral. This is borne out by recent
patch-clamp studies on Xenopus oocytes expressing amine
transporters (Sonders et al., 1997). In addition to the
transporter-mediated ion flux resulting from ion cotrans-
port (in the case of SERT plus countertransport), uncou-
pled ion fluxes also occur, which have prompted the idea
that amine transporters have an external and internal gate.
This could explain the observed currents: (1) a substrate-
mediated current, blocked by nonsubstrate inhibitors, and
(2) a constitutive leak current, blocked by both substrates
and nonsubstrate inhibitors; for both currents, the external
and internal gates need to be transiently and simultane-
ously open. See also: Transgenic Xenopus production
It should be mentioned here that interference with ion
gradients can cause amine transporters to function in the
reversed mode (i.e. effluxing amine from the cytoplasm to
the exterior). It is still a matter of debate as to whether, or
how much, this mode contributes to physiological (ex-
tra)synaptic function. It is clear that reversed transport can
play an important role under extreme conditions that dis-
turb normal ion gradients, such as ischaemia, or in the
action of releasing stimulants such as amphetamine (see the
section Transporters as targets for drug action). In the case
of reversed transport induced by substrates such as am-
phetamine, the process is more complicated than mere ex-
change and likely involves intracellular Na+ made
available by substrate influx (Khoshbouei et al., 2003).
See also: Stroke
Amine substrate is translocated together with Na+ and
Cl . For the noradrenaline transporter, Na+ probably
2
binds before noradrenaline and Cl2, whereas there is no
3
 Amine Transporters
agreement on the order of binding of Na+, Cl2 and do-
pamine to the dopamine transporter. All of this does not
necessarily imply that Na+ or Cl2 is required for the bind-
ing of substrate in the first step of the translocation cycle
(Figure 3). Li et al. (2002) showed that Na+ is neither
required nor stimulatory for dopamine, amphetamine,
tyramine and octopamine binding to the human DAT, in
sharp contrast to the stimulatory effect of Na+ on cocaine
analogue binding.
Regulation of Amine Transporters
Regulation of transporter function can be acute (seconds
to minutes) or long term (hours or days). Acute regulation
can be brought about by changes in ion or substrate gra-
dients, by cell-surface distribution, by reversible interac-
tions of the transporter with regulatory proteins, by
posttranslational modification of the transporter inserted
in the plasma membrane, and by perturbation of the mem-
brane environment surrounding the transporter. Long-
term regulation of transporter function can occur as a re-
sult of altered gene expression, or posttranslational mod-
ification of the DAT before insertion in the plasma
membrane. Whereas several hours are needed to progress
from transcribed mRNA to transporters functionally
present in the plasma membrane, even more time is need-
ed to move transport proteins to nerve endings distal to the
translation machinery. Glycosylation could be viewed as a
long-term regulation process, occurring as a process tightly
linked to protein synthesis in the endoplasmic reticulum
(ER) and near the Golgi compartments. Phosphorylation
is an example of acute regulation, and so is the perturba-
tion of transporter environment by the lipid messenger
arachidonic acid. See also: Axonal transport and the
neuronal cytoskeleton; Regulation by covalent modifica-
tion; Translation control by RNA
In general, glycosylation can be N-, O- or C-terminus-
linked. In the amine transporters, glycosylation is N-linked
to Asn. Mutants of the dopamine, noradrenaline and se-
rotonin transporter that lack the N-glycosylation consen-
sus sites, and therefore exist in the unglycosylated form
have also been studied. These mutants are impaired in tar-
geting of the transporter to the plasma membrane and sta-
bility of membrane-resident transporters (Melikian et al.,
1996; Torres et al., 2003). For the NAT and SERT, un-
glycosylated transporter recognize substrates and blockers
normally; for the DAT, more work is needed to arrive at
this conclusion (see Li et al., 2004).
Consensus sites are present in amine transporters for
phosphorylation by protein kinase C (PKC), protein ki-
nase A (PKA), and Ca2+ –calmodulin-dependent protein
kinase II (CaM-KII) (Figure 2). There is evidence for the
implication of these kinases in transporter function. In
particular, activation of PKC reduces the uptake activity of
4
the DAT, NAT and SERT by reducing their presence on
the cell surface. Furthermore, it is known that PKC acti-
vators enhance the phosphorylation state of the DAT and
SERT, with the recent detailed information on N-terminal
serine residues in the DAT targeted by PKC. For both
transporters, PKC consensus sites do not appear to be in-
volved, because mutants with sites that have been removed
still respond with the same decrease in uptake to PKC ac-
tivation. It is beginning to be more probable that phos-
phorylation is involved in the case of proteins associated
with the amine transporters, such as syntaxin 1A, protein
phosphatase, (PICK-1), or Hic-5.
In general, arachidonic acid decreases neurotransmitter
transport, such as for glutamate (by the excitatory amino
acid transporter subtype 1 (EAAT-1)), GABA and glycine.
The amine transporter for dopamine can also be either
inhibited by arachidonic acid, or enhanced, depending on
experimental conditions. Increased uptake by arachidonic
acid has also been reported for the glutamate transporter
subtype EAAT-2. In the case of the dopamine transporter,
inhibition by arachidonic acid does not underlie the effect
of PKC activation, which could be thought to enhance the
activity of mitogen-activated protein kinase, which in turn
phosphorylates phospholipase A2, generating arachidonic
acid. Rather than arachidonic acid being the precursor for
eicosanoids, there is evidence for a role of endogenous
arachidonic acid itself in the transporter effects, with a role
for cis-unsaturation, i.e. folding or bending of its hydro-
carbon tails. Other recent examples of acute regulation of
amine transporters are the effects described as a result
of the presence of transporter blockers or substrates, or of
presynaptic receptor ligands (Gulley and Zahniser, 2003).
See also: Amino acid transporters; Arachidonic acid;
Glycine as a neurotransmitter
Animal models have provided many examples of the
downregulation of a number of noradrenaline and
serotonin transporters by long-term treatment with anti-
depressant drugs. In addition, regulation of dopamine
transporter numbers has been reported to occur in selected
brain regions by using treatment regimens of dopamine
transporter inhibitors.
Involvement of Amine Transporters in
Psychiatric Disorders
Dopamine (DA) is implicated in attention deficit/hyperac-
tivity disorder (ADHD), schizophrenia, drug abuse, Par-
kinson disease and Tourette syndrome (TS). The strongest
evidence so far for the linkage or association of DAT po-
lymorphisms is in the area of ADHD, with a possible in-
fluence of the 10-repeat allele of the human DAT 3’ variable
number of tandem repeats (VNTRs) (see Hahn and Blak-
ely, 2002). Other polymorphisms are known, such as Taq1
polymorphisms, intron 8 VNTRs and single-nucleotide

polymorphisms (SNPs), but more work is needed to es-
tablish their involvement in diseases in which DA is im-
plicated. Some evidence links sequences in the 3’ region of
the human DAT gene other than the 3’ VNTRs to bipolar
disorder. Furthermore, in a study of Australian Caucasian
Parkinson disease patients, a 10-fold increased risk was
associated with the rare 11-repeat allele. Although DA is
strongly implicated in schizophrenia, supported by simi-
larity between the phenotype of DAT-knockout mice and
the symptomology of schizophrenia, a relationship be-
tween human DAT polymorphism and schizophrenia has
not been established. See also: Attention deficit–hyper-
activity disorder; Linkage and crossing over; Parkinson
disease; Schizophrenia
Noradrenergic pathways in the brain are implicated in
arousal, response to stress and the related phenomenon
depression. In addition, the NAT is important in the aut-
onomic system with cardiovascular implications. Two
types of human NAT (hNAT) polymorphisms, Taq1 po-
lymorphisms and SNPs have been reported. Taq1, the first
identified hNAT polymorphism, has not yet been linked
with bipolar disorder and major depression. Among the
many SNPs, no associations between disease states, in-
cluding depression, have been found as yet, except for the
correlation between ‘orthostatic intolerance’, a disorder
characterized by an increase in heart rate upon standing,
without concomittant hypotension, and the A457P variant
in transmembrane domain 9 (Hahn and Blakely, 2002).
Serotonin plays a role in a wide range of psychiatric
disorders, such as affective disorders, suicide and aggres-
sive-impulsive behaviour. Depression has been associated
with a decrease in neuronal serotonin uptake by the SERT.
One of the known SERT polymorphisms, the SERT long
promoter region repeat element deletion polymorphism (5-
HTTLPR), is associated with enhanced serotonin uptake
and may also be associated with impulsive violent behav-
iour, including violence co-morbid with alcohol depend-
ence or suicide attempts. A VNTR in intron 3 (formerly
designated intron 2 prior to the identification of novel up-
stream intron) (the 9-repeat allele) has been associated with
depression in one study, but conflicting results have been
reported in other studies. See also: Alcoholism; Autism;
Serotonin
MPTP and Parkinson Disease
In the early 1980s, several heroin addicts inadvertently self-
administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine (MPTP) and developed acute Parkinson disease.
MPTP is now commonly used in the mouse and monkey
model of Parkinson disease. MPTP is converted by brain
monoamine oxidase B to MPP+ (1-methyl-4-pyridinium
ion), which is taken up by the DAT into dopaminergic
terminals, where it can exert its neurotoxic effect probably
Amine Transporters
by interfering with mitochondrial function. The neurotox-
ic effect of MPTP correlates with the expression level of the
DAT. In this context, the action of the DAT in the plasma
membrane appears to be opposed by the amine transporter
in the storage vesicle, which is of the vesicular monoamine
transporter 2 (VMAT-2) subtype in vesicles in mono-
aminergic neurons. Thus, VMAT-2 mediates the uptake of
MPP+ into vesicles, thereby diminishing cytoplasmic con-
centrations of the neurotoxic MPP+. The property of
VMAT to transport MPP+ was actually used to clone rat
VMAT-1 by testing the ability of cDNA to make Chinese
hamster ovary (CHO) cells resistant to MPP+ by trans-
porting it into intracellular acidic compartments away
from the cytoplasm. In addition, heterozygous VMAT-2
knockout mice display enhanced MPTP toxicity, in agree-
ment with a protective role of vesicular uptake of MPP+.
See also: Basal ganglia and the regulation of movement;
Dopamine; Knockout and knock-in animals; Parkinson
disease
Transporters as Targets for Drug Action
Amine transporters in plasma membranes are targets for
various drugs. The DAT is inhibited potently, and selec-
tively, in comparison with the NAT and SERT, by the
diphenyl-piperazine derivatives GBR 12909 and 12935. In
animal models, these compounds have stimulatory effects
similar to cocaine although, depending on dose, GBR
12909 can also partially antagonize the action of cocaine.
Although cocaine is not selective for the DAT among the
amine transporters, there is substantial evidence implicat-
ing DAT blockade in the centrally stimulating activity of
cocaine (Ritz et al., 1987), especially in the nucleus accum-
bens, where nerve terminals of the mesolimbic dopamine
system terminate. Knockout mice that lack the DAT show
enhanced locomotion as a result of continuously higher
levels of extracellular dopamine, but do not respond to
cocaine with additional locomotor activation, as expected
if the DAT were required for this effect (Giros et al., 1996).
In contrast, these mice still self-administer cocaine, pre-
senting a paradox if one accepts the requirement of the
DAT for the rewarding activity of cocaine (Rocha et al.,
1998). Possible explanations for the latter observation in-
clude a compensatory effect occurring during development
without the DAT from birth, the involvement of blockade
by cocaine of the NAT, which clears dopamine in the pre-
frontal cortex where the density of DAT is low in normal
mice, or the involvement of NAT in the nucleus accumbens
in DAT knockout mice. It is also possible that SERT takes
on a role in the effect of cocaine in DAT knockouts, with
combined DAT/NAT knockouts still displaying cocaine-
conditioned place preference. See also: Cocaine and
amphetamines
5
 Amine Transporters
Amphetamine is another psychostimulant drug that is
generally believed to require the presence of the DAT.
Unlike cocaine, amphetamine is a substrate for the trans-
porter, and can enter nerve terminals through the trans-
porter, in addition to sheer membrane penetration based
on its lipophilicity. Amphetamine is known to induce re-
versed dopamine transport, and the lack of this phenom-
enon in DAT knockout mice supports the requirement of
the transporter in this process. In addition, amphetamine
depletes vesicular dopamine, leading to reduced exocytotic
dopamine release.
Molecular analysis by site-directed mutagenesis has
shown an important role for Asp79 in the first transmem-
brane domain in both dopamine uptake and cocaine-like
binding, and for Ser356 and Ser359 in the seventh trans-
membrane domain, more in dopamine uptake than co-
caine-like binding (see Chen and Reith (2002) for a review
on molecular structure–function studies in amine trans-
porters). Experiments on the corresponding serines in
NAT argue against a direct hydrogen bond interaction
between catechol substrate and the two serines. There is
evidence that differences between the rat and human dop-
amine transporter in recognizing MPP+ may be explained
by the fact that residue 533 is Tyr in the rat and Phe in the
human. See also: Mutagenesis: site-specific
The NAT is potently and selectively blocked by many
antidepressant drugs such as desipramine, nisoxetine, nor-
triptyline and nomifensine. Molecular analyses have been
conducted based on chimaeras between the NAT and DAT
in which similar sequence domains were exchanged. Trans-
membrane domains 5–8 were shown to be important for
conferring sensitivity to various inhibitors such as desipra-
mine and GBR 12935, domains 1–3 to substrates and the
region between domains 11 and 12 to MPP+. Similar
NAT–DAT chimaeras were examined in a different study
indicating substrate recognition by transmembrane do-
mains 9–12. Site-directed mutagenesis studies are providing
additional insights. Cysteine residues have been studied
with membrane impermeant methane thiosulfonate
(MTS)-type sulfhydryl reagents in modified DAT, devoid
of a number of endogenous reactive cysteines residues that
were subsequently placed back, one by one. Reaction of
Cys90 or Cys306 with MTS enhanced DA uptake and co-
caine analogue binding by inducing a conformational
change (Ferrer and Javitch, 1998). Conformational states
of DAT favourable to DA binding may differ from those
favourable to cocaine binding because rat DAT mutants
such as F76A, P101A, F155A and W406A display in-
creased DA affinity but decreased cocaine analogue affin-
ity. Furthermore, Na+-induced conformational changes
favour cocaine analogue binding but not DA binding in
human DAT mutants W84L and D313N (Chen et al.,
2002). In the human NAT, mutants F316C, V356S and
G400L (replacement residues are those in the correspond-
ing position in DAT) showed decreased potency of the tri-
cyclics desipramine and nortriptyline, but normal potency
6
of cocaine. The latter indicates that the loss of cocaine ac-
tivity in NAT–DAT chimaeras with DAT sequences in the
transmembrane 5–8 region was caused by interdomain in-
teractions rather than the loss of specific cocaine interaction
points. Among human NAT variants, G478S displayed
defective norepinephrine uptake of potential interest in
diseases characterized by disturbed catecholamine levels,
such as heart failure and a number of mental diseases.
See also: Hallucinogenic drugs; Primary protein and nucleic
acid three-dimensional structure databases
Although NAT blockade can result in antidepressant
activity, there are other drugs for the treatment of depres-
sion that rely on selective blockade of the SERT, exempli-
fied by fluoxetine, citalopram and sertraline. Studies on
chimaeras between the SERT and the NAT support the
conclusion that the amino or carboxy tail is not directly
involved in substrate or blocker recognition. In addition,
results on chimaeras between the human and rat SERT
indicate that a region in or near transmembrane domain 12
interacts with imipramine. Site-directed mutagenesis
showed that Phe586 in the 11th transmembrane domain
is responsible for the enhanced antidepressant sensitivity in
the human SERT, as compared with Val586 in the rat
SERT. Changing Tyr95 in transmembrane domain 1 to
Phe, as in the Drosophila SERT, shifted the citalopram and
mazindol potencies to those observed in Drosophila SERT.
In a different study, Ile172 and Tyr176 in transmembrane
domain 3 were found to be in proximity to the binding site
for serotonin and cocaine (Chen et al., 1997). D98 in hu-
man SERT, corresponding to D79 in rat DAT, may di-
rectly interact with the positively charged amine group of
substrate, unlike the situation for DAT. In transmembrane
7 of human SERT, positions 368, 372, 376, 380 and 384 fall
on a stripe that runs at an angle down one side of the
predicted a-helix of transmembrane 7, and mutations at
these locations cause reduced substrate uptake. In a study
of currents mediated by the human and rat SERT, Thr490
in the rat transporter was implicated in transport-mediated
current, possibly by its location on the external gate in the
gating model for amine transporters (see Cao et al., 1998 in
Further Reading). Ser545 in rat SERT has also been pro-
posed to be part of the external gate.
The above results from mutagenesis studies are just a
selection of mutations published so far. It will be a formi-
dable challenge to place all attempted mutations in a larger
context. It is likely that different inhibitors will interact
with the same transporter in different ways, whereas, con-
versely, the same inhibitor can interact with different
transporters in a different manner.
Summary
Initial pharmacological and biochemical work identified
separate transport systems for dopamine, noradrenaline

and serotonin, which mediate the uptake of amines into
nerve endings against their concentration gradient. The
individual transport proteins have been cloned and fur-
ther characterized as plasma membrane proteins with 12
transmembrane domains; these proteins can form oligo-
mers. The amine transporters carry sites for glycosylation
and phosphorylation which play a role in trafficking
processes regulating the number of membrane-resident
transporters. Transmembrane ion gradients, maintained
by Na+/K+ ATPase drive uptake, are additionally elect-
rogenic in the case of dopamine and noradrenaline such
that the negative interior potential promotes uptake.
Furthermore, channel properties of transporters are re-
sponsible for ion fluxes in addition to those strictly cou-
pled to substrate fluxes. Amine transporters in plasma
membranes are targets for various drugs such as psy-
chostimulants and antidepressants, and the dopamine
transporter has been implicated in the uptake of neuro-
toxins causing Parkinson disease. Associations between a
number of psychiatric disorders and polymorphisms in
amine transporters have been reported that do not alter
the transporter proteins themselves but could be involved
in the regulation of their expression.
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Enzymology 296: 1–730.
Cao Y, Li M, Mager S and Lester HA (1998) Amino acid residues that
control pH modulation of transport-associated current in mammalian
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influence dopamine uptake and cocaine analog recognition. Molecular
Pharmacology 56: 434–447.
Lin Z, Wang W and Uhl GR (2000) Dopamine transporter tryptophan
mutants highlight candidate dopamine- and cocaine-selective do-
mains. Molecular Pharmacology 58: 1581–1592.
Reith MEA (ed.) (2002) Neurotransmitter Transporters: Structure,
Function and Regulation, 2nd edn. Totowa, NJ: Humana Press.
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Reith MEA (guest ed.) (2003) Transporters as targets for drugs and
endogenous compounds. European Journal of Pharmacology, (Special
Issue) Vol. 479, Nos. 1–3.
Robinson MB (2002) Regulated trafficking of neurotransmitter trans-
porters: common notes but different melodies. Journal of Neuro-
chemistry 80: 1–11.
Roubert C, Cox PJ, Brüss M et al. (2001) Determination of residues
in the norepinephrine transporter that are critical for tricyclic
antidepressant affinity. Journal of Biological Chemistry 276: 8254–
8260.