Juan Zhen

LeuT-Desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake

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 angstroms 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.

Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia

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.

Clinical and molecular characterisation of hereditary dopamine transporter deficiency syndrome: an observational cohort and experimental study

Summary Background Dopamine 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. Methods 11 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. Findings Children 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. Interpretation Dopamine 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. Funding Birmingham Childrens Hospital Research Foundation, Birth Defects Foundation Newlife, Action Medical Research, US National Institutes of Health, Wellchild, and the Wellcome Trust.

Dopamine transporter deficiency syndrome: phenotypic spectrum from infancy to adulthood.

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.

Interaction of cocaine-, benztropine-, and GBR12909-like compounds with wild-type and mutant human dopamine transporters: molecular features that differentially determine antagonist-binding properties

The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine’s behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [3H]CFT binding to wild‐type, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2‐benzhydrylpiperidine) exhibited a cocaine‐like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine‐like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3α stereochemistry tended to exhibit benztropine‐like binding, whereas those with 3β stereochemistry were more cocaine‐like. Our results point to the importance of specific molecular features – most notably the presence of a diphenylmethoxy moiety – in determining a compound’s binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine‐like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor–transporter interaction could lead to the development of DAT inhibitors with differential clinical utility.

Chronic food restriction and dopamine transporter function in rat striatum.

The present communication reports on DA uptake in rat striatum in a model of chronic food restriction. The K(m) for DA uptake was unaltered, but the V(max) was reduced by 32%, not supporting the idea that the enhanced behavioral sensitivity to cocaine or d-amphetamine upon chronic food restriction is due to a greater density of DAT at the plasma membrane for drug interaction. Chronic food restriction did not alter the potency of cocaine or D-amphetamine in inhibiting DA uptake in the striatum, suggesting that the enhanced behavioral sensitivity to these drugs upon chronic food restriction is not due to their enhanced affinity for DAT. These results point to factors other than DAT density or affinity underlying the sensitized response to psychostimulants in food restriction.

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Development of (S)-N6-(2-(4-(isoquinolin-1-yl)piperazin-1-yl)ethyl)-N6-propyl-4,5,6,7-tetrahydrobenzo[d]-thiazole-2,6-diamine and its analogue as a D3 receptor preferring agonist: potent in vivo activity in Parkinson's disease animal models.

Here we report structure-activity relationship study of a novel hybrid series of compounds where structural alteration of aromatic hydrophobic moieties connected to the piperazine ring and bioisosteric replacement of the aromatic tetralin moieties were carried out. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (K(i)). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. SAR results identified compound (-)-24c (D-301) as one of the lead molecules with preferential agonist activity for D3 receptor (EC(50) (GTP gamma S); D3 = 0.52 nM; D2/D3 (EC(50)): 223). Compounds (-)-24b and (-)-24c exhibited potent radical scavenging activity. The two lead compounds, (-)-24b and (-)-24c, exhibited high in vivo activity in two Parkinsons disease (PD) animal models, reserpinized rat model and 6-OHDA induced unilaterally lesioned rat model. Future studies will explore potential use of these compounds in the neuroprotective therapy for PD.

Importance of cholesterol in dopamine transporter function

The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid‐raft localized DATs compared with non‐raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT‐mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode voltammetry (RDEV). Treatment with methyl‐β‐cyclodextrin (mβCD), which effectively depletes total membrane cholesterol––uniformly affecting cholesterol–DAT interactions in both raft and non‐raft membrane domains––reduced both DA uptake and efflux rate. In contrast, disruption of raft‐localized DAT by cholesterol chelation with nystatin had no effect, arguing against a vital role for raft‐localized DAT in substrate uptake or efflux. Supranormal repletion of cholesterol‐depleted cells with the analog desmosterol, a non‐raft promoting sterol, was as effective as cholesterol itself in restoring transport rates. Further studies with Zn2+ and the conformationally biased W84L DAT mutant supported the idea that cholesterol is important for maintaining the outward‐facing DAT with normal rates of conformational interconversions. Collectively, these results point to a role for direct cholesterol–DAT interactions in regulating DAT function.

Substrate and drug binding sites in LeuT

LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine, and dopamine. The original crystal structure of LeuT shows a primary leucine-binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters.