?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
Homozygous loss-of-function mutations
in the gene encoding the dopamine
transporter are associated
with infantile parkinsonism-dystonia
Manju A. Kurian,1,2 Juan Zhen,3 Shu-Yuan Cheng,3 Yan Li,3 Santosh R. Mordekar,4 Philip Jardine,5
Neil V. Morgan,1 Esther Meyer,1 Louise Tee,1 Shanaz Pasha,1 Evangeline Wassmer,2
Simon J.R. Heales,6 Paul Gissen,1 Maarten E.A. Reith,3,7 and Eamonn R. Maher1,8
1Department of Medical and Molecular Genetics, University of Birmingham School of Medicine, Institute of Biomedical Research, Birmingham,
United Kingdom. 2Department of Paediatric Neurology, Birmingham Children’s Hospital, Birmingham, United Kingdom.
3Department of Psychiatry, Millhauser Laboratories, New York University School of Medicine, New York, New York, USA.
4Department of Paediatric Neurology, Sheffield Children’s Hospital, Sheffield, United Kingdom. 5Department of Paediatric Neurology, Bristol Children’s Hospital,
Bristol, United Kingdom. 6Neurometabolic Unit, National Hospital, and Department of Chemical Pathology, Great Ormond Street Hospital, London,
United Kingdom. 7Department of Pharmacology, New York University School of Medicine, New York, New York, USA.
8West Midlands Regional Genetics Service, Birmingham Women’s Hospital, Birmingham, United Kingdom.
Parkinson disease is the second most common neurodegenera-
tive disorder after Alzheimer disease and affects approximately 1%
of the population over 50 years of age. Although most cases of
Parkinson disease are sporadic, a number of inherited disorders
can be associated with parkinsonism. Molecular genetic investi-
gation of these cases can provide insights into the pathogenesis
of movement disorders and/or neurodegeneration. The subgroup
of patients who present with early-onset disease are of particular
interest. Infantile parkinsonism-dystonia (IPD) is a severe neuro-
logical syndrome that usually presents in early infancy with hypo-
kinetic parkinsonism (1). Subsequently, a complex movement dis-
order with dystonia develops in association with axial hypotonia
and limb hypertonicity. The condition is severe and can be fatal.
IPD is rare but is often underdiagnosed, as the clinical presenta-
tion can mimic certain types of cerebral palsy. Elucidating the
molecular basis of IPD might provide insights into disease patho-
genesis and allow earlier diagnosis.
In order to define the molecular basis of IPD, we used autozy-
gosity mapping techniques to perform molecular genetic inves-
tigations in 2 consanguineous families. The disease locus was
mapped to chromosome 5p15.3. We demonstrated different germ-
line SLC6A3 gene mutations in both kindreds. Functional stud-
ies revealed that both mutations led to a reduction in the level of
mature dopamine transporter (DAT).
Two consanguineous families with IPD were investigated. Family 1
was a large consanguineous kindred of Pakistani origin (Figure 1A)
with 2 affected children (patients 1 and 2), who were first cousins.
Family 2 (Figure 1B) was of mixed European descent with 1 affected
child (patient 3). The diagnosis of IPD was based on characteristic
clinical features and cerebrospinal fluid (CSF) neurotransmitter
studies (Table 1). In brief, following a normal pregnancy and birth,
all children had neonatal irritability and feeding difficulties. Par-
kinsonism-dystonia heralded disease onset in early infancy and was
rapidly followed by the development of pyramidal tract features.
Two patients (patients 1 and 3) were initially misdiagnosed with
cerebral palsy. On examination at ages 6–12 months, all patients
had features of parkinsonism, dystonia, and pyramidal tract signs
with evidence of global developmental delay. CSF biogenic amine
metabolite studies in affected patients revealed markedly elevated
concentrations of homovanillic acid (HVA; with normal 5-hydroxy-
indoleacetic acid levels). MRI brain scans did not reveal structural
abnormalities in any of the patients. Formal neuropsychiatric
assessments to determine whether the children showed any evi-
dence of cognitive impairment were not conducted. All patients
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Nonstandard?abbreviations?used: CFT, 2β-carbomethoxy-3β-(4-fluorophenyl)-
tropane; CSF, cerebrospinal fluid; DAT, dopamine transporter; DBS, deep brain
stimulator; hDAT, human DAT; HVA, homovanillic acid; IPD, infantile parkinson-
Citation?for?this?article: J. Clin. Invest. 119:1595–1603 (2009). doi:10.1172/JCI39060.
Related Commentary, page 1455
1596?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
showed a poor clinical response to multiple therapeutic agents.
CSF HVA levels did not normalize after L-dopa or tetrabenazine
therapy. Insertion of a deep-brain stimulator device in patient 3
resulted in some symptomatic improvement of dystonia and rigid-
ity. All children showed a progressive disease course with worsen-
ing symptoms of parkinsonism, dystonia, and hypertonicity. None
of the affected children showed evidence of psychiatric or behav-
ioral/conduct disorders. Detailed neurological examination of all
the children’s parents (ages 21 to 37 years) and siblings (ages 1 to 9
years) revealed no neurological abnormalities. Additionally, none of
the unaffected family members (including siblings, obligate carrier
parents, parental siblings, and grandparents) were known to have a
neuropsychiatric or age-related movement disorder.
In the light of the diagnosis of autosomal recessive IPD and
parental consanguinity, we undertook an autozygosity mapping
study in family 1. Initially, genome-wide SNP genotyping (using
the Affymetrix 250K SNP microarray) was performed in patients
1 and 2 from family 1. In these 2 affected cousins, 4 regions of
extended homozygosity (>2 Mb) were detected (on chromosomes
5 [2.3 Mb], 7 [6 Mb], 9 [41 Mb], and 14 [9 Mb]). These regions were
then further analyzed using polymorphic microsatellite markers
(~0.5–2 Mb apart) mapping to these regions of interest. Linkage to
the candidate regions on chromosomes 7 (4 markers), 9 (6 mark-
ers), and 14 (5 markers) was either excluded by segregation of the
microsatellite marker alleles or the finding of heterozygous alleles
in affected individuals (data not shown). On chromosome 5, the
Family trees of consanguineous kindreds. (A) Family 1 and (B) family 2 kinship. Children affected with IPD are indicated by black shading. Circles
indicate females; squares indicate males; diamonds indicate undisclosed gender.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
2 children shared a common homozygous haplotype of 201 SNPs
from 1 to 2,373,003 bp, thereby defining the margins of the homo-
zygous region, while the unaffected sibling of patient 1 demon-
strated SNP heterozygosity in this region (Figure 2). Genotyping
of the family with 4 microsatellite markers (D5S2488, D5S1981,
D5S2005, and D5678) within the interval revealed an identical
homozygous haplotype in the 2 affected children, consistent with
linkage to this region (Figure 3). Subsequent microsatellite marker
analysis of family 2 was also consistent with disease linkage to this
region (Figure 4). Multipoint linkage analysis gave a maximum lod
score of 4.76 (family 1, 3.29; family 2, 1.46).
After establishing the region on chromosome 5 as the loca-
tion of the IPD gene, we examined the 39 genes within the region
for a candidate IPD gene. SLC6A3 (located at chromosome 5;
1,445,909–1,498,538 bp) encoded a DAT, and mutation analy-
sis of SLC6A3 was initiated. A
homozygous putative missense
mutation c.1103T>A (p.L368Q)
(Figure 5) was detected at exon
8 in patients 1 and 2. Follow-
ing identification of the puta-
tive mutation, SLC6A3 muta-
tion testing was performed in
family 2, and a homozygous
putative mutation in exon
9, c.1184C>T (p.P395L), was
detected in patient 3 (Figure
5). Both mutations segregated
with IPD disease status in the
families, and neither mutation
was detected in an extensive
analysis of ethnically matched
control chromosomes (544
alleles from Asian individu-
als and 438 alleles from indi-
viduals of mixed European
descent). Sequence alignment
showed Leu368 and Pro395 to
be highly conserved through-
out the species (Figure 6). Both
residues (particularly Leu368)
were largely conserved in other
human SLC6 transporters, sug-
gesting that they are important
for transporter function but
perhaps not for dopamine-spe-
cific transporter properties.
To determine the effects of
the L368Q and P395L muta-
tions, mutant human DAT
(hDAT) proteins were tran-
siently expressed in HEK293
cells and their transport activ-
ity compared with that of
wild-type hDAT (see Methods
and Table 2) (2–4). In experi-
ments carried out in parallel,
wild-type hDAT showed nor-
mal transport activity, whereas
L368Q and P395L hDAT were
devoid of uptake activity. The binding affinity of the cocaine ana-
log [3H]2β-carbomethoxy-3β-(4-fluorophenyl)-tropane ([3H]CFT)
was near normal in the mutants (30–36 nM, compared with 16
nM in wild-type hDAT; see Table 2). In contrast, the potency
of dopamine in inhibiting cocaine analog binding was greatly
reduced in L368Q hDAT (KI increased by an order of magnitude),
whereas the potency of dopamine in P395L hDAT was close to
that of wild type (1). Thus, both mutations were loss-of-function
mutations with respect to the capability of DAT to translocate
its substrate, dopamine, in conjunction with a loss of apparent
binding affinity of dopamine in the case of one of the mutations.
Maximal binding of [3H]CFT to cells, primarily representing sur-
face binding (5), indicated appreciable expression of DAT in both
mutants, although L368Q showed a reduction that was statisti-
cally significant (Table 2).
Phenotypic data for patients with IPD
Pregnancy and birth
Early course in infancy
Onset of symptoms, mo
Pyramidal tract signs
Eye movement disorder
Features at clinical examination
Reduced facial expression
Pyramidal tract signs in limbs
Axial hypotonia/head lag
Global developmental delay
Features at eye examination
Saccade initiation failure
CSF HVA concentrationA
CSF 5-HIAA concentrationB
CSF HVA:5-HIAA ratioC
Urine HVA:creatine ratioD
Response to medicationF
Response to deep brain stimulator
Progression of symptoms
Mixed European descent
Parents 2nd cousinsParents - 1st cousins;
patient 1 and patient 2
are first cousins
(mothers are sisters)
Father and maternal
feeding difficultiesfeeding difficulties
–, Absent; +, present; ++, severe; 5-HIAA, 5-hydroxyindoleacetic acid; ND, not determined. AReference range,
154–867 nmol/l. BReference range, 89–367 nmol/l. CReference range, 1.0–3.7. DReference range, 2–15 μmol
HVA/mmol creatine. EReference range, 93–630 mIU/l. FTherapeutic agents included levodopa, carbidopa, tetra-
benazine, diazepam, carbamazepine, baclofen, 5-hydroxytryptophan and trihexyphenidyl.
1598?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
To investigate whether the observed reductions in dopamine
transport were related to disordered production, location, or
function of the mutant proteins, immunoblotting was per-
formed. Analysis of whole cell lysates demonstrated equivalent
levels of immature (55-kDa) DAT for wild-type and P395L, but
there was some reduction for L368Q (total protein loading, as
judged by β-actin, was equivalent for wild-type, L368Q, and
P395L hDAT). However both mutant proteins demonstrated a
profound reduction in mature (85-kDa) DAT compared with
wild-type DAT. Analysis of surface (biotinylated DAT) showed a
similar pattern (Figure 7).
To date, inherited infantile parkinsonism syndromes are usually
associated with reduced CSF dopamine metabolites and enzyme
defects in neurotransmitter pathways (6). Here we describe a dis-
tinct type of IPD, with characteristic clinical and unique biochem-
ical features, caused by mutations in SLC6A3. This early-onset
complex movement disorder can be mistaken for cerebral palsy
and is, to our knowledge, the first instance in which patients with
parkinsonian features have been linked to an inherited disorder of
The pre-synaptic DAT, encoded by SLC6A3, mediates the active
reuptake of extraneuronal dopamine and is a principal regulator of
the amplitude and duration of dopaminergic action at presynaptic
and postsynaptic receptors. DAT is exclusively expressed in dopa-
mine neurons with significantly higher levels of DAT expression in
cells of the substantia nigra pars compacta, substantia nigra pars
lateralis, and ventral tegmental area (7). Dopamine is transported
inward against its concentration gradient using the driving force of
the sodium gradient across the plasma membrane. The transport
process of dopamine uptake involves translocation of dopamine as
well as 2 sodium ions and 1 chloride ion across the cell membrane
(8). The Na+/K+ pump thus has a crucial role in generating the elec-
Localization of the disease locus on chromosome 5 on a 250K Affyme-
trix SNP array. SNP array data for patient 1 (VII:1) and patient 2 (VII:3)
and the unaffected sibling of patient 1 (VII:2). Patients 1 and 2 dis-
played a region of SNP homozygosity from 0–2.3 Mb. The unaffected
sibling did not display this common SNP homozygosity. Dark blue, AA
homozygous SNP call; mid-blue, BB homozygous SNP call; light blue,
no call; white, AB heterozygous call.
Microsatellite marker linkage analysis on chromo-
some 5 in family 1. Affected individuals are indicated
by black symbols. SLC6A3 gene is located at 5p15.3.
Microsatellite markers are positioned according to
physical distance (measured in Mb). Haplotypes for
these markers are shown, and the disease-associ-
ated haplotypes are boxed in blue. Green, pink, and
yellow boxes indicate non–disease-associated hap-
lotypes. The 2 affected children shared a common
homozygous haplotype in this region. N, novel mic-
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
trochemical gradient across the cell membrane and inherited muta-
tions in the gene for the Na+/K+-ATPase α3 subunit (ATP1A3) are
associated with autosomal dominant rapid-onset parkinsonism-
dystonia (ROPD) (9). Loss-of-function ATP1A3 mutations might
thus also possibly indirectly affect DAT function.
Based on the crystal structure of the bacterial transporter analog
LeuT (10), the primary substrate site and the sodium binding sites
in DAT can be modeled deep in the protein interior, where unwind-
ing of the transmembrane helices 1 and 6 allows extensive interac-
tions among protein residues in transmembrane domains 1, 3, 6, 7,
and 8 and substrate or sodium (11). A secondary substrate site has
been proposed to be located toward the outside of the transporter
(12) in an extracellular vestibule where tricyclic antidepressants
also bind in LeuT (2, 13), above the extracellular gate, which, in
turn, lies between the 2 substrate sites. Among the positions of
the 2 IPD-associated missense mutations reported here, Leu368 in
hDAT was in transmembrane domain 7 toward the exterior, many
helix turns above the residue (Asn353) that presumably interacts
with sodium (Figure 8). Leu368 is 1 helix turn under Met371 and
has been shown to be conformationally sensitive to dopamine
transport (14). Of the 2 helical portions of extracellular loop 4,
EL4b, the position of the second missense mutation, Pro395, is
in the helical portion closer to transmembrane 8. Pro395 lies just
1 helix turn above Ala399, a residue that, like Met371, points out-
ward from the entire transporter structure and thus is easily acces-
sible to solvent bathing the outside of the protein (14). The inter-
action between a sulfhydryl reagent from the outside and position
399 was not blocked in the presence of dopamine, whereas the
interaction at the 371 position was highly sensitive to dopamine,
which caused conformational changes, burying the residue away
from solvent exposure. In consonance, the missense mutation of
Pro395 (above 399) had no impact on dopamine binding affinity,
whereas the missense mutation of Leu368 (under 371) appeared to
interfere with conformational changes occurring with dopamine
binding, such that dopamine’s apparent affinity was strongly
reduced. Hence, despite the different impact of the 2 missense
mutations on dopamine binding affinity, the location of the muta-
tions and the fact that the mutations completely impaired trans-
port of dopamine suggested that both mutations might interfere
with conformational changes required during transport. However,
a major contributor to reducing dopamine transport appears to
be the marked reduction in mature (glycosylated) 85-kDa DAT
expression. It has previously been demonstrated that immature
non-glycosylated (55-kDa) DAT does not transport dopamine
as efficiently as mature 85-kDa DAT (15). Hence a reduction in
mature DAT would undoubtedly impair dopamine transport. It is
known that glycosylation patterns vary with the cellular environ-
ment, which is dependent upon cell type and physiological state of
the cell in which the glycoprotein is made, and so analysis of brain
DAT glycosylation in DAT IPD cases is of considerable interest.
Dopamine mediates a wide array of physiological functions,
including locomotion, cognitive processes, neuroendocrine secre-
tion, and the control of motivated behaviors including emotion,
affect, and reward mechanisms. DAT is the site of action of drugs
such as cocaine and methylphenidate (which inhibit DAT-medi-
ated dopamine reuptake) and amphetamines (16). Dysfunctional
dopamine homeostasis has been implicated in a wide number of
neurological and neuropsychiatric conditions (17, 18), and the
observation that DAT homozygous-null mice exhibit a hyper-
dopaminergic phenotype with prominent hyperactivity (19) has
prompted extensive genetic investigations of SLC6A3 in human
neuropsychiatric disorders. Mazei-Robison et al. reported a rare
coding variant (A559V) in 2 male siblings with attention deficit
hyperactivity disorder (20). The A559V mutant demonstrated
increased dopamine efflux (which characteristically is associ-
ated with amphetamine-like psychostimulants), and so different
genetic alterations in SLC6A3 may affect different facets of DAT
function and cause distinct phenotypes. Although the phenotypic
features seen in IPD are not evident in the established knockout
mouse model, a more highly developed extrapyramidal system in
primates may result in differences in attributes of DAT dysfunc-
tion in mice and humans.
Although IPD is a rare condition, it is likely underdiagnosed
and may mimic mixed forms of cerebral palsy, in which pyrami-
Microsatellite marker analysis on chromosome 5 in family 2.
The black symbol indicates the affected patient. Microsatel-
lite markers are positioned according to physical distance
(measured in Mb). Haplotypes for these markers are shown,
and the disease-associated haplotypes are boxed in red.
Orange and blue boxes indicate non–disease-associated
haplotypes. Microsatellite markers show evidence of segre-
gation with disease status in this family.
1600?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
dal features and dystonia coexist. CSF dopamine metabolites are
abnormal in IPD and might be used to identify patients with vari-
ant phenotypes for SLC6A3 mutation analysis. As the spectrum
of SLC6A3 mutations expand, specific genotype-phenotype cor-
relations may emerge. In addition, genetic variants in SLC6A3 may
influence susceptibility to other movement disorders such as late-
onset parkinsonism and dystonic side effects of anti-parkinsonism
drugs or neuroleptics.
Finally, molecular characterization of SLC6A3-associated IPD
may provide a basis for the development of effective therapies.
Current therapies do not biochemically normalize HVA levels or
clinically improve patient symptomatology. L-dopa therapy may
be beneficial in other infantile parkinsonism syndromes (such
as tyrosine hydroxylase deficiency) with reduced dopamine pro-
duction. In IPD patients with SLC6A3 mutations, defective DAT
function and loss of dopamine reuptake may lead to depletion
of neuronal dopamine stores. In fact, data from mice with tar-
geted genetic deletion of dopamine, serotonin, and norepineph-
rine transporters provide further evidence that these monoamine
transporters have an important role in monoamine homeostasis.
SLC6A3 mutations in patients 1 and 3. The alignments of SLC6A3 nucleotides c.1091–c.1116 (A–C) and c.1168–c.1197 (D–F) are shown.
B illustrates the c.1103T>A mutation in family 1. E illustrates the c.1184C>T mutation in family 2.
Conservation of the mutated DAT1
residues L368 and P395. Five rep-
resentative vertebrate sequences
are aligned. L368 and P395 are
shown in red. Residues matching
the consensus sequence are in
gray; those not matching the con-
sensus sequence are in white.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
Such mice show reduced extracellular monoamine clearance rate,
decreased amplitude of stimulated monoamine release, elevated
basal extracellular monoamine levels, decreased monoamine tis-
sue storage, increased monoamine synthesis rate, and disrupted
autoreceptor function (21). Additionally, the reduced reuptake of
dopamine by DAT increases the dopamine in the extraneuronal
space (as shown by increased HVA in the CSF), which might result
in secondary downregulation of postsynaptic dopamine receptor
expression. Certainly in the aforementioned monoamine trans-
porter knockout mouse models, there is evidence of postsynaptic
receptor downregulation (21). Our patients with DAT IPD dem-
onstrated a poor response to L-dopa, suggesting that therapeutic
attempts to normalize brain dopamine levels in DAT IPD might
be insufficient to improve function, perhaps because of the com-
plex role of DAT in the fine regulation of amplitude and duration
of dopaminergic transmission. Although short-term DAT inhibi-
tion appears to potentiate L-dopa therapy in Parkinson disease
(22), our experience with DAT IPD suggests that this might not
be maintained in the long term. The moderate therapeutic suc-
cess of DBS in patient 3 may be attributable to the hypothesized
role of such targeted neuronal high-frequency stimulation in the
modulation of dopamine, glutamate, and GABA neurotransmitter
systems within the basal ganglia (23). Such selective neuronal acti-
vation and suppression as well as changes in network synchrony
may thus result in alleviation of parkinsonian-dystonic symptoms.
In the future, trials of DBS as well as partial dopamine receptor
agonists/antagonists could be useful for medically ameliorating
DAT-associated movement disorders.
Family members and control subjects provided written informed con-
sent under a research protocol approved by the South Birmingham Local
Research Ethics Committee. All 3 affected children with IPD were clinically
assessed by a pediatric neurologist. Formal neurological examination of all
the parents and unaffected siblings was also undertaken.
Molecular genetic studies
DNA extraction. DNA was extracted from peripheral lymphocytes using
Genome-wide scan. In order to identify common regions of shared homo-
zygosity in affected individuals, a genome-wide scan using the Affymetrix
250K SNP microarray was initially
undertaken. DNA was amplified and
hybridized to the Affymetrix 250K
SNP chips according to the manufac-
Linkage analysis. All significant
regions of common homozygosity
identified on genome-wide scan were
further investigated using standard
polymorphic microsatellite markers
PCR amplification of genomic DNA
samples, amplified fragments were
genotyped using an ABI PRISM 3730
Genetic Analyzer and analyzed using
GeneMapper software. Scored geno-
types were assembled as haplotypes and analyzed for evidence of linkage.
Gene sequencing. Mutation analysis of SLC6A3 (DAT1) was undertaken by
direct sequencing. Primer pairs were designed for exon-specific PCR ampli-
fication of the 14 translated exons of the SLC6A3 gene (Table 3). At the
purification stage, amplified products were either treated with Exosap or
gel-extracted (Qiagen). Sequencing reactions were performed using BigDye
Terminator v3.1 Cycle Sequencing kits and run on an ABI PRISM 3730
DNA Analyzer (Applied Biosystems). DNA sequences were viewed and ana-
lyzed using Chromas software.
Functional analysis of mutant DAT proteins
Mutant constructs of L368Q hDAT and P395L hDAT were prepared
from wild-type pCIN4-hDAT (a gift from Jonathan A. Javitch, Columbia
University, New York, New York, USA) as the template for PCR using the
QuikChange site-directed mutagenesis kit (Stratagene) with some modi-
fications. Briefly, 50 ng of DNA template (pCIN4-hDAT) was mixed with
a primer and its complementary primer (100 ng each), 1 μl dNTP mix,
2.5 μl of 10× reaction buffer, and 1.25 U of PfuTurbo DNA polymerase in
a final volume of 25 μl. The PCR conditions included an initial denatur-
ation cycle (1 min, 95°C); 18 cycles of denaturation (30 s, 95°C), anneal-
Dopamine transport and cocaine analog binding by wild-type, L368Q, and P395L hDAT
?Vmax (pmol/min/mg protein)
?Bmax (pmol/mg protein)
Inhibition of [3H]CFT binding by dopamine
0.249 ± 0.029 (4)
1.19 ± 0.20 (4)
15.9 ± 1.9 (5)
2.14 ± 0.65 (5)
35.6 ± 8.2 (3)
0.384 ± 0.110B (3)
30.2 ± 6.5 (5)
0.962 ± 0.144 (5)
6.58 ± 0.53 (4) 52.9 ± 1.7B (3) 3.35 ± 0.65 (3)
Bmax, maximal binding. Data are mean ± SEM. The number of experiments performed for each group is indi-
cated in parentheses. ANo transport activity was detectable above non-specific uptake. BP < 0.05 compared
with wild type, by 1-way ANOVA followed by Dunnett multiple comparisons.
Expression of the L368Q hDAT and P395L hDAT in transiently
transfected HEK293 cells via immunoblotting analysis. Cells were
transiently transfected with wild-type, L368Q, and P395L hDAT and
reagent only. In each panel, the same amount of protein was loaded
in all lanes. Biotinylated (surface) protein and total lysate were probed
with anti-DAT antibody for detection of the relative expression level of
wild-type and mutant DAT protein. Anti–β-actin antibody showed the
relative equivalent loading of total protein.
1602?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
ing (1 min, 55°C), and extension (16 min, 68°C); a final extension cycle
(10 min, 68°C); and termination at 4°C. PCR products were digested
with 10 U of DpnI at 37°C for 1 h to remove the parental strands. The
digested DNA mixture was transformed into E. coli XL-1 blue supercom-
petent cells by heat shock. Mutagenesis was verified by DNA sequence
analysis (by the DNA sequencing facility at the Skirball Institute of Bio-
molecular Medicine, New York University). The following primers and
their complementary primers (IDT Inc.) were used: L368Q, 5′-GTCGT-
GTTCAGCTTTCAGGGATACATGGCCCAG-3′; P395L, 5′-GATCTTCAT-
Culturing and transient transfection of HEK293 cells with Lipo-
fectamine 2000, was carried out as described previously (2). Uptake of
[3H]dopamine into hDAT-expressing cells was measured for 5 minutes
at 21°C, as in our previous study (2), with high sodium, low potassium,
glucose and tropolone containing buffer (3). Nonspecific uptake was
defined by 1 μM CFT. For measurement of cocaine analog binding, cells
were incubated with 4 nM [3H]CFT (PerkinElmer) for 20 minutes at
21°C in 200 μl of the same buffer used for the uptake assay. For satura-
tion analysis, increasing concentrations of nonradioactive CFT (0.1–100
nM) were included in assay. The procedures were described previously
(4). Nonspecific binding was defined with 1 μM CFT. The Km and Vmax
of dopamine uptake and the Kd and maximal binding (Bmax) of [3H]CFT
binding were estimated with nonlinear regression fitting using Radlig
software (KELL program; Biosoft). The equilibrium dissociation KI of
[3H]CFT binding was computed using the Cheng-Prusoff equation (4)
from the concentration inhibiting binding by 50%, as estimated using
logistic fitting of data by the ORIGIN software (OriginLab Co.). Com-
parisons of the 2 mutants with wild-type DAT were performed using
1-way ANOVA followed by the Dunnett multiple comparisons test. Sta-
tistical significance was considered at P < 0.05.
DAT surface protein preparation by biotinylation
and Western blotting
Cells transiently expressing wild-type hDAT, L368Q hDAT, and P395L
hDAT and mock cells were processed as described previously (24), with
minor changes. Briefly, the cells were washed with cold PBS and incu-
bated with sulfo-NHS-SS-biotin (1 mg/ml PBS; Pierce Biotechnology)
for 60 minutes at 4°C, followed by incubation with 50 mM glycine in
PBS for 20 minutes and extensive washing. The washed cells were lysed
in mammalian protein extraction reagents supplemented with a protein
Primers used to sequence coding regions of the SLC6A3 gene
of DAT topology based on LeuT
structure. Twelve transmembrane
domains are shown with helically
unwound regions in the first and
sixth domain; extracellular and
intracellular loops include helical
portions (e2, e3, e4a, e4b, and i1,
i5, respectively) (10, 11). Leu368
(L; red), subject to missense muta-
tion, is shown at the top of trans-
membrane domain 7, one helix turn
under Met371 (M; blue). Pro395
(P; red), also subject to missense
mutation, is shown in the e4b por-
tion of extracellular loop 4, one helix
turn above Ala399 (A; blue). In the
3-dimensional structure, sodium
ions interact not only with residues
of transmembrane domains 1, 6,
and 8, but also with transmembrane
domain 7 (10).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 6 June 2009
inhibitor cocktail for 10 minutes at 21°C followed by 60 minutes at 4°C
with vortexing at 5-minute intervals. The lysate samples were centri-
fuged at 14,000 g for 15 minutes to remove cell debris. The biotinylated
proteins were separated with immobilized monomeric NeutrAvidin
(Pierce Biotechnology) and eluted with SDS-PAGE sample buffer. The
total lysates and biotinylated proteins were resolved on 4%–12% Bis-
tris NuPAGE gels (Invitrogen). DAT was probed with polyclonal anti-
DAT antibody (0.3 μg/ml) against the C-terminal of DAT (Millipore),
followed by HRP-conjugated goat anti-rabbit antibody. Polyclonal
anti–β-actin antibody (Sigma-Aldrich) was used as an internal control
for loading. The transporter signal was visualized using SuperSignal
West Dura Extended Duration chemiluminescence substrate solution
We thank the Birmingham Children’s Hospital Research Foundation,
NewLife, Action Medical Research, the Wellcome Trust, WellChild,
and the NIH for financial support. We also thank Robert Surtees.
Received for publication February 27, 2009, and accepted in revised
form April 8, 2009.
Address correspondence to: Eamonn R. Maher, Department of
Medical and Molecular Genetics, Institute of Biomedical Research,
Birmingham University, Edgbaston, Birmingham B15 2TT, United
Kingdom. Phone: 00-44-121-627-2741; Fax: 00-44-121-414-2538;
1. Assmann, B.E., et al. 2004. Infantile Parkinsonism-
dystonia and elevated dopamine metabolites in
CSF. Neurology. 62:1872–1874.
2. Zhou, Z., et al. 2007. LeuT-desipramine structure
reveals how antidepressants block neurotransmit-
ter reuptake. Science. 317:1390–1393.
3. Chen, N., Rickey, J., Berfield, J.L., and Reith, M.E.A.
2004. Aspartate 345 of the dopamine transporter
is critical for conformational changes in substrate
translocation and cocaine binding. J. Biol. Chem.
4. Chen, N., and Reith, M.E. 2007. Substrates and
inhibitors display different sensitivity to expres-
sion level of the dopamine transporter in heterolo-
gously expressing cells. J. Neurochem.?101:377–388.
5. Chen, N., Zhen, J., and Reith, M.E. 2004. Mutation
of Trp84 and Asp313 of the dopamine transporter
reveals similar mode of binding interaction for
GBR12909 and benztropine as opposed to cocaine.
J. Neurochem. 89:853–864.
6. Pearl, P.L., Taylor, J.L., Trzcinski, S., and Sokohl, A.
2007.The paediatric neurotransmitter disorders.
J. Child Neurol. 22:606–616.
7. Storch, A., Ludolph, A.C., and Schwarz, J. 2004.
Dopamine transporter: involvement in selective
dopaminergic neurotoxicity and degeneration.
J. Neural Transm. 111:1267–1286.
8. McElvain, J.S., and Schenk, J.O. 1992. A multisub-
strate mechanism of striatal dopamine uptake
and its inhibition by cocaine. Biochem. Pharmacol.
9. de Carvalho Aguiar, P., et al. 2004. Mutations in the
Na+/K+ -ATPase alpha3 gene ATP1A3 are associ-
ated with rapid-onset dystonia parkinsonism. Neu-
10. Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., and
Gouaux, E. 2005. Crystal structure of a bacterial
homologue of Na+/Cl––dependent neurotransmit-
ter transporters. Nature. 437:215–223.
11. Beuming, T., Shi, L., Javitch, J.A., and Weinstein, H.
2006. A comprehensive structure-based alignment
of prokaryotic and eukaryotic neurotransmitter/
Na+ symporters (NSS) aids in the use of the LeuT
structure to probe NSS structure and function.
Mol. Pharmacol. 70:1630–1642.
12. Shi, L., Quick, M., Zhao, Y., Weinstein, H., and
Javitch, J.A. 2008. The mechanism of a neurotrans-
mitter:sodium symporter–inward release of Na+
and substrate is triggered by substrate in a second
binding site. Mol. Cell. 30:667–677.
13. Singh, S.K., Yamashita, A., and Gouaux, E. 2007.
Antidepressant binding site in a bacterial homo-
logue of neurotransmitter transporters. Nature.
14. Norregaard, L., Loland, C.J., and Gether, U. 2005.
Evidence for distinct sodium-, dopamine-, and
cocaine-dependent conformational changes in
transmembrane segments 7 and 8 of the dopamine
transporter.?J. Biol. Chem. 278:30587–30596.
15. Li, L.B., et al. 2004. The role of N-glycosylation in
function and surface trafficking of the human dopa-
mine transporter. J. Biol. Chem. 279:21012–21020.
16. Bannon, M.J. 2005. The dopamine transporter: role
in neurotoxicity and human disease. Toxicol. Appl.
17. Mehler-Wex, C., Riederer, P., and Gerlach, M. 2006.
Dopaminergic dysbalance in distinct basal ganglia
neurocircuits: implications for the pathophysi-
ology of Parkinson’s disease, schizophrenia and
attention deficit hyperactivity disorder. Neurotox.
18. Mazei-Robinson, M.S., and Blakely, R.D. 2006.
ADHD and the dopamine transporter: are there
reasons to pay attention? Handb. Exp. Pharmacol.
19. Giros, B., Jaber, M., Jones, S.R., Wightman, R.M., and
Caron, M.G. 1996. Hyperlocomotion and indiffer-
ence to cocaine and amphetamine in mice lacking
the dopamine transporter.?Nature. 379:606–612.
20. Mazei-Robison, M.S., et al. 2008. Anomalous
dopamine release associated with a human
dopamine transporter coding variant. J. Neurosci.
21. Gainetdinov, R.R., and Caron, M.G. 2003. Mono-
maine transporters: from genes to behavior. Annu.
Rev. Pharmacol. Toxicol. 43:261–284.
22. Nutt, J.G., Carter, J.H., and Sexton, G.J. 2004. The
dopamine transporter: importance in Parkinson’s
disease. Ann. Neurol. 55:766–773.
23. McIntyre, C.C., Savasta, M., Walter, B.L., and Vitek,
J.L. 2004. How does deep brain stimulation work?
Present understanding and future questions.
J. Clin. Neurophysiol. 21:40–50.
24. Chen, N., and Reith, M.E. 2008. Substrates dissoci-
ate dopamine transporter oligomers. J. Neurochem.