Tricyclic antidepressants exert their pharmacological effect -inhibiting the reuptake of serotonin, norepinephrine and dopamine -by directly blocking neurotransmitter transporters (SERT, NET and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 Å of the bacterial leucine transporter (LeuT), a homolog of SERT, NET and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters. Na + /Cl − -dependent neurotransmitter transporters for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) in the presynaptic plasma membrane terminate neuronal signal transmission in the central nervous system through a reuptake mechanism (1-6). These systems have been shown to modulate mood, emotion, sleep and appetite (7). Depression, arguably the most prevalent psychiatric disorder, is directly associated with perturbation of serotonergic neurotransmission (8, 9), and drugs blocking serotonin reuptake have been used successfully for its treatment. One class of these drugs, tricyclic antidepressants (TCAs) such as desipramine and imipramine, binds to serotonin and norepinephrine transporters with affinities of nanomolar to tens of nanomolar concentrations and blocks transport activity (10). The response rate of patients to TCAs is typically 60-70% (11). More recently, highly selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac) have also been developed and are increasingly prescribed to treat depression (12). The molecular The human SERT, DAT and NET proteins all belong to a family of transporters for amino acids and their derivatives, the Neurotransmitter:Sodium Symporter (NSS) family (2)(3)(4)(5)14). Whilst the dopamine transporters from human, bovine or rat are inhibited by TCAs at a K i of micromolar concentrations, the DAT proteins from C. elegans (15) and D. melanogaster (16) are inhibited by TCAs at a K i of nanomolar and sub-micromolar concentrations, respectively (17). As bacterial NSS proteins share up to 30 % sequence identity with human SERT and NET as well as worm and fly DATs, we hypothesized that bacterial NSS proteins also possess high binding affinity to TCAs and could provide opportunities for studying proteindrug interactions. We therefore chose a bacterial NSS protein, the leucine transporter (LeuT) from Aquifex aeolicus, to study the molecular mechanism of neurotransmitter transporter binding to TCAs (18). LeuT shares 2...

Sertraline and fluoxetine are selective serotonin reuptake inhibitors (SSRIs) widely-prescribed to treat depression. They exert their effects by inhibiting the presynaptic plasma membrane serotonin transporter (SERT). All SSRIs possess at specific positions halogen atoms, which are key determinants for the drugs’ specificity for SERT. For the SERT protein, however, the structural basis of its specificity for SSRIs is poorly understood. Here we report the crystal structures of LeuT, a bacterial SERT homolog, in complex with sertraline, R-fluoxetine or S-fluoxetine. The SSRI halogens all bind to exactly the same pocket within LeuT. Mutation at this halogen-binding pocket (HBP) in SERT dramatically reduces the transporter's affinity for SSRIs but not for tricyclic antidepressants. Conversely, when the only non-conserved HBP residue in both norepinephrine and dopamine transporters is mutated into that found in SERT, their affinities for all the three SSRIs increase uniformly. Thus, the specificity of SERT for SSRIs is dependent largely on interaction of the drug halogens with the protein's halogen-binding pocket.

SummaryBackgroundDopamine transporter deficiency syndrome is the first identified parkinsonian disorder caused by genetic alterations of the dopamine transporter. We describe a cohort of children with mutations in the gene encoding the dopamine transporter (SLC6A3) with the aim to improve clinical and molecular characterisation, reduce diagnostic delay and misdiagnosis, and provide insights into the pathophysiological mechanisms.Methods11 children with a biochemical profile suggestive of dopamine transporter deficiency syndrome were enrolled from seven paediatric neurology centres in the UK, Germany, and the USA from February, 2009, and studied until June, 2010. The syndrome was characterised by detailed clinical phenotyping, biochemical and neuroradiological studies, and SLC6A3 mutation analysis. Mutant constructs of human dopamine transporter were used for in-vitro functional analysis of dopamine uptake and cocaine-analogue binding.FindingsChildren presented in infancy (median age 2·5 months, range 0·5–7) with either hyperkinesia (n=5), parkinsonism (n=4), or a mixed hyperkinetic and hypokinetic movement disorder (n=2). Seven children had been initially misdiagnosed with cerebral palsy. During childhood, patients developed severe parkinsonism-dystonia associated with an eye movement disorder and pyramidal tract features. All children had raised ratios of homovanillic acid to 5-hydroxyindoleacetic acid in cerebrospinal fluid, of range 5·0–13·2 (normal range 1·3–4·0). Homozygous or compound heterozygous SLC6A3 mutations were detected in all cases. Loss of function in all missense variants was recorded from in-vitro functional studies, and was supported by the findings of single photon emission CT DaTSCAN imaging in one patient, which showed complete loss of dopamine transporter activity in the basal nuclei.InterpretationDopamine transporter deficiency syndrome is a newly recognised, autosomal recessive disorder related to impaired dopamine transporter function. Careful characterisation of patients with this disorder should provide novel insights into the complex role of dopamine homoeostasis in human disease, and understanding of the pathophysiology could help to drive drug development.FundingBirmingham Children's Hospital Research Foundation, Birth Defects Foundation Newlife, Action Medical Research, US National Institutes of Health, Wellchild, and the Wellcome Trust.

Dopamine transporter deficiency syndrome is an SLC6A3-related progressive infantile-onset parkinsonism-dystonia that mimics cerebral palsy. Ng et al. describe clinical features and molecular findings in a new cohort of patients. They report infants with classical disease, as well as young adults manifesting as atypical juvenile-onset parkinsonism-dystonia, thereby expanding the disease spectrum.

Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.