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1.  Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates 
Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [3H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., 4 doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, two weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [3H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.
doi:10.1038/npp.2013.331
PMCID: PMC3988539  PMID: 24287719
serotonin (5-HT) release; 5-HT depletion; 5-HT transporter (SERT); SERT substrate; SERT-mediated current
2.  PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein 
Nature chemical biology  2014;10(7):582-589.
Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.
doi:10.1038/nchembio.1545
PMCID: PMC4062427  PMID: 24880859
dopamine; transporter; amphetamine; Drosophila melanogaster; phosphatidylinositol 4,5-bisphosphate
3.  In Vivo Amphetamine Action is Contingent on αCaMKII 
Neuropsychopharmacology  2014;39(11):2681-2693.
Addiction to psychostimulants (ie, amphetamines and cocaine) imposes a major socioeconomic burden. Prevention and treatment represent unmet medical needs, which may be addressed, if the mechanisms underlying psychostimulant action are understood. Cocaine acts as a blocker at the transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET), but amphetamines are substrates that do not only block the uptake of monoamines but also induce substrate efflux by promoting reverse transport. Reverse transport has been a focus of research for decades but its mechanistic basis still remains enigmatic. Recently, transporter-interacting proteins were found to regulate amphetamine-triggered reverse transport: calmodulin kinase IIα (αCaMKII) is a prominent example, because it binds the carboxyl terminus of DAT, phosphorylates its amino terminus, and supports amphetamine-induced substrate efflux in vitro. Here, we investigated whether, in vivo, the action of amphetamine was contingent on the presence of αCaMKII by recording the behavioral and neurochemical effects of amphetamine. Measurement of dopamine efflux in the dorsal striatum by microdialysis revealed that amphetamine induced less dopamine efflux in mice lacking αCaMKII. Consistent with this observation, the acute locomotor responses to amphetamine were also significantly blunted in αCaMKII-deficient mice. In addition, while the rewarding properties of amphetamine were preserved in αCaMKII-deficient mice, their behavioral sensitization to amphetamine was markedly reduced. Our findings demonstrate that amphetamine requires the presence of αCaMKII to elicit a full-fledged effect on DAT in vivo: αCaMKII does not only support acute amphetamine-induced dopamine efflux but is also important in shaping the chronic response to amphetamine.
doi:10.1038/npp.2014.124
PMCID: PMC4207348  PMID: 24871545
4.  A Cytosolic Relay of Heat Shock Proteins HSP70-1A and HSP90β Monitors the Folding Trajectory of the Serotonin Transporter* 
The Journal of Biological Chemistry  2014;289(42):28987-29000.
Background: Naturally occurring mutations in solute carrier 6 (SLC6) family members impair folding of these transporters.
Results: Folding-deficient SERT mutants bound heat shock protein (HSP)70-1A, HSP90β, and co-chaperones. Noribogaine synergized with HSP inhibitors in rescuing these SERT mutants.
Conclusion: Folding of SERT is assisted by a cytosolic HSP relay.
Significance: The folding trajectory of SERT is relevant to folding diseases arising from mutated SLC6 transporters.
Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90β. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90β by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90β. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.
doi:10.1074/jbc.M114.595090
PMCID: PMC4200255  PMID: 25202009
Glycosylation; Heat Shock Protein (HSP); Heat Shock Protein 90 (Hsp90); Serotonin; Serotonin Transporter; Trafficking; Transporter
5.  Structure and Regulatory Interactions of the Cytoplasmic Terminal Domains of Serotonin Transporter 
Biochemistry  2014;53(33):5444-5460.
Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.
doi:10.1021/bi500637f
PMCID: PMC4147951  PMID: 25093911
6.  Additive Gene–Environment Effects on Hippocampal Structure in Healthy Humans 
The Journal of Neuroscience  2014;34(30):9917-9926.
Hippocampal volume loss has been related to chronic stress as well as genetic factors. Although genetic and environmental variables affecting hippocampal volume have extensively been studied and related to mental illness, limited evidence is available with respect to G × E interactions on hippocampal volume. The present MRI study investigated interaction effects on hippocampal volume between three well-studied functional genetic variants (COMT Val158Met, BDNF Val66Met, 5-HTTLPR) associated with hippocampal volume and a measure of environmental adversity (life events questionnaire) in a large sample of healthy humans (n = 153). All three variants showed significant interactions with environmental adversity with respect to hippocampal volume. Observed effects were additive by nature and driven by both recent as well as early life events. A consecutive analysis of hippocampal subfields revealed a spatially distinct profile for each genetic variant suggesting a specific role of 5-HTTLPR for the subiculum, BDNF Val66Met for CA4/dentate gyrus, and COMT Val158Met for CA2/3 volume changes. The present study underscores the importance of G × E interactions as determinants of hippocampal volume, which is crucial for the neurobiological understanding of stress-related conditions, such as mood disorders or post-traumatic stress disorder (PTSD).
doi:10.1523/JNEUROSCI.3113-13.2014
PMCID: PMC4107408  PMID: 25057194
COMT; SLC6A4; BDNF; MRI; hippocampus; stress
7.  Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters☆ 
Neurochemistry International  2014;73(100):32-41.
Highlights
•We quantified adulterants in street drugs sold as cocaine.•We analyzed effects of the most common adulterant levamisole, on neurotransmitter transporters.•Differences in the selectivity of levamisole can be explained by homology modelling and docking.•Aminorex, a metabolite of levamisole, modulates neurotransmitter transporters directly.•Depending on the transporter, aminorex acts as a blocker or as a releaser.
Psychostimulants such as amphetamine and cocaine are illicitly used drugs that act on neurotransmitter transporters for dopamine, serotonin or norepinephrine. These drugs can by themselves already cause severe neurotoxicity. However, an additional health threat arises from adulterant substances which are added to the illicit compound without declaration. One of the most frequently added adulterants in street drugs sold as cocaine is the anthelmintic drug levamisole. We tested the effects of levamisole on neurotransmitter transporters heterologously expressed in HEK293 cells. Levamisole was 100 and 300-fold less potent than cocaine in blocking norepinephrine and dopamine uptake, and had only very low affinity for the serotonin transporter. In addition, levamisole did not trigger any appreciable substrate efflux. Because levamisole and cocaine are frequently co-administered, we searched for possible allosteric effects; at 30 μM, a concentration at which levamisole displayed already mild effects on norepinephrine transport it did not enhance the inhibitory action of cocaine. Levamisole is metabolized to aminorex, a formerly marketed anorectic drug, which is classified as an amphetamine-like substance. We examined the uptake-inhibitory and efflux-eliciting properties of aminorex and found it to exert strong effects on all three neurotransmitter transporters in a manner similar to amphetamine. We therefore conclude that while the adulterant levamisole itself has only moderate effects on neurotransmitter transporters, its metabolite aminorex may exert distinct psychostimulant effects by itself. Given that the half-time of levamisole and aminorex exceeds that of cocaine, it may be safe to conclude that after the cocaine effect “fades out” the levamisole/aminorex effect “kicks in”.
doi:10.1016/j.neuint.2013.11.010
PMCID: PMC4077236  PMID: 24296074
SERT, serotonin transporter; NET, norepinephrine transporter; DAT, dopamine transporter; 5-HT, serotonin; DA, dopamine; KHB, Krebs–Ringer–HEPES buffer; HPLC, high performance liquid chromatography; LC–MS, liquid chromatography–mass spectrometry; Levamisole; Aminorex; Neurotransmitter transporter; Cocaine; Adulterant
8.  Missense dopamine transporter mutations associate with adult parkinsonism and ADHD 
The Journal of Clinical Investigation  2014;124(7):3107-3120.
Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.
doi:10.1172/JCI73778
PMCID: PMC4071392  PMID: 24911152
9.  Evidence for a Role of Transporter-Mediated Currents in the Depletion of Brain Serotonin Induced by Serotonin Transporter Substrates 
Neuropsychopharmacology  2014;39(6):1355-1365.
Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [3H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [3H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.
doi:10.1038/npp.2013.331
PMCID: PMC3988539  PMID: 24287719
5-HT depletion; neurotransmitters; psychopharmacology; psychostimulants; serotonin; serotonin (5-HT) release; serotonin (5-HT) transporter; SERT substrate; SERT-mediated current; toxicity; serotonin (5-HT) release; 5-HT depletion; 5-HT transporter (SERT); SERT substrate; SERT-mediated current
10.  Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity 
PLoS ONE  2014;9(3):e92543.
Background
The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence.
Methods
A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax.
Results
The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity.
Conclusion
This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation.
doi:10.1371/journal.pone.0092543
PMCID: PMC3965432  PMID: 24667541
11.  Powerful Cocaine-Like Actions of 3,4-Methylenedioxypyrovalerone (MDPV), a Principal Constituent of Psychoactive ‘Bath Salts' Products 
Neuropsychopharmacology  2012;38(4):552-562.
The abuse of psychoactive ‘bath salts' containing cathinones such as 3,4-methylenedioxypyrovalerone (MDPV) is a growing public health concern, yet little is known about their pharmacology. Here, we evaluated the effects of MDPV and related drugs using molecular, cellular, and whole-animal methods. In vitro transporter assays were performed in rat brain synaptosomes and in cells expressing human transporters, while clearance of endogenous dopamine was measured by fast-scan cyclic voltammetry in mouse striatal slices. Assessments of in vivo neurochemistry, locomotor activity, and cardiovascular parameters were carried out in rats. We found that MDPV blocks uptake of [3H]dopamine (IC50=4.1 nℳ) and [3H]norepinephrine (IC50=26 nℳ) with high potency but has weak effects on uptake of [3H]serotonin (IC50=3349 nℳ). In contrast to other psychoactive cathinones (eg, mephedrone), MDPV is not a transporter substrate. The clearance of endogenous dopamine is inhibited by MDPV and cocaine in a similar manner, but MDPV displays greater potency and efficacy. Consistent with in vitro findings, MDPV (0.1–0.3 mg/kg, intravenous) increases extracellular concentrations of dopamine in the nucleus accumbens. Additionally, MDPV (0.1–3.0 mg/kg, subcutaneous) is at least 10 times more potent than cocaine at producing locomotor activation, tachycardia, and hypertension in rats. Our data show that MDPV is a monoamine transporter blocker with increased potency and selectivity for catecholamines when compared with cocaine. The robust stimulation of dopamine transmission by MDPV predicts serious potential for abuse and may provide a mechanism to explain the adverse effects observed in humans taking high doses of ‘bath salts' preparations.
doi:10.1038/npp.2012.204
PMCID: PMC3572453  PMID: 23072836
addiction & Substance Abuse; blocker; catecholamines; designer drugs; neuropharmacology; Psychostimulants; releaser; transporter; designer drug; dopamine; cathinone; monoamine transporter; uptake blocker
12.  A Combined Approach Using Transporter-Flux Assays and Mass Spectrometry to Examine Psychostimulant Street Drugs of Unknown Content 
ACS Chemical Neuroscience  2012;4(1):182-190.
The illicit consumption of psychoactive compounds may cause short and long-term health problems and addiction. This is also true for amphetamines and cocaine, which target monoamine transporters. In the recent past, an increasing number of new compounds with amphetamine-like structure such as mephedrone or 3,4-methylenedioxypyrovalerone (MDPV) entered the market of illicit drugs. Subtle structural changes circumvent legal restrictions placed on the parent compound. These novel drugs are effectively marketed “designer drugs” (also called “research chemicals”) without any knowledge of the underlying pharmacology, the potential harm or a registration of the manufacturing process. Accordingly new entrants and their byproducts are identified postmarketing by chemical analysis and their pharmacological properties inferred by comparison to compounds of known structure. However, such a heuristic approach fails, if the structures diverge substantially from a known derivative. In addition, the understanding of structure–activity relations is too rudimentary to predict detailed pharmacological activity. Here, we tested a combined approach by examining the composition of street drugs using mass spectrometry and by assessing the functional activity of their constituents at the neuronal transporters for dopamine, serotonin, and norepinephrine. We show that this approach is superior to mere chemical analysis in recognizing novel and potentially harmful street drugs.
doi:10.1021/cn3001763
PMCID: PMC3547486  PMID: 23336057
Amphetamine; bath-salts; mass spectrometry; combo; psychostimulants; 2C-B; MDPV
13.  Pharmacological examination of trifluoromethyl ring-substituted methcathinone analogs 
European journal of pharmacology  2012;699(0):180-187.
Cathinones are a class of drugs used to treat various medical conditions including depression, obesity, substance abuse, and muscle spasms. Some “designer” cathinones, such as methcathinone, mephedrone, and methylone, are used nonclinically for their stimulant or entactogenic properties. Given the recent rise in nonmedical use of designer cathinones, we aimed to improve understanding of cathinone pharmacology by investigating analogs of methcathinone with a CF3 substituent at the 2-, 3-, or 4-position of the phenyl ring (TFMAPs). We compared the TFMAPs with methcathinone for effects on monoamine uptake transporter function in vitro and in vivo, and for effects on locomotor activity in rats. At the serotonin transporter (SERT), 3-TFMAP and 4–TFMAP were 10-fold more potent than methcathinone as uptake inhibitors and as releasing agents, but 2-TFMAP was both a weak uptake inhibitor and releaser. At the norepinephrine and dopamine transporters (NET and DAT), all TFMAP isomers were less potent than methcathinone as uptake inhibitors and releasers. In vivo, 4-TFMAP released 5-HT, but not dopamine, in rat nucleus accumbens and did not affect locomotor activity, whereas methcathinone increased both 5-HT and dopamine and produced locomotor stimulation. These experiments reveal that TFMAPs are substrates for the monoamine transporters and that phenyl ring substitution at the 3- or 4-position increases potency at SERT but decreases potency at NET and DAT, resulting in selectivity for SERT. The TFMAPs might have a therapeutic value for a variety of medical and psychiatric conditions and may have lower abuse liability compared to methcathinone due to their decreased DAT activity.
doi:10.1016/j.ejphar.2012.11.008
PMCID: PMC3656655  PMID: 23178523
Bath salts; Cathinone; Designer drug; Dopamine; Mephedrone; Methylone; Monoamine transporter; Norepinephrine; Serotonin
14.  Membrane Localization is Critical for Activation of the PICK1 BAR Domain 
Traffic (Copenhagen, Denmark)  2008;9(8):1327-1343.
The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C-terminus transferred to the single transmembrane protein Tac or the dopamine transporter C-terminus transferred to Tac. In contrast, transfer of the GluR2 C-terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain-dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N-terminal myristoylation site produced BAR domain-dependent, but ligand-independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain-dependent and linker-dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane-binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment.
doi:10.1111/j.1600-0854.2008.00761.x
PMCID: PMC3622726  PMID: 18466293
BAR domains; PDZ domains; protein–lipid interactions; receptors; transporters
16.  Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model 
PLoS Computational Biology  2013;9(2):e1002909.
The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.
Author Summary
The dopamine transporter (DAT) regulates dopaminergic neurotransmission in the brain and is implicated in numerous human disease states. DAT is unique among the monoamine neurotransmitter transporter family because its substrate transport is inhibited by extracellular zinc. DAT homology models rely upon the crystal structure of LeuT solved in 2005. LeuT and DAT share a relatively low overall sequence identity of 22%. In addition, the length of the second extracellular loop of DAT exceeds that of LeuT by 21 residues. The zinc binding site cannot be directly modeled from the LeuT template alone because of these differences. Current available homology models of DAT focused on substrate or inhibitor binding rather than on the second extracellular loop. We exploited the specificity of the zinc binding site to build and calibrate a DAT homology model of the complete transmembrane domain. Our model predicted that the zinc binding site in DAT consists of four zinc co-ordinating residues rather than three that had been previously identified. We verified this hypothesis by site-directed mutagenesis and uptake inhibition studies.
doi:10.1371/journal.pcbi.1002909
PMCID: PMC3578762  PMID: 23436987
17.  Switching the Clientele 
The Journal of Biological Chemistry  2013;288(8):5330-5341.
Background: The serotonin transporter (SERT) relies exclusively on SEC24 paralog C for its ER export.
Results: A lysine to tyrosine mutation in the C terminus of SERT switches its preference from SEC24C to SEC24D.
Conclusion: The position +2 from the RI/RL/KL export motif determines SEC24 paralog requirement.
Significance: The role of SEC24C in ER export of neurotransmitter transporters is relevant to psychiatric disorders, e.g., bipolar disease and depression.
The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif (607RI608 in SERT). Substituting Lys610 with tyrosine, the corresponding residue found in the noradrenaline and dopamine transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine transporter-1a, the betaine/GABA transporter and the GABA transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter transporters.
doi:10.1074/jbc.M112.408237
PMCID: PMC3581386  PMID: 23288844
Dopamine Transporters; Endoplasmic Reticulum (ER); Membrane Transport; Monoamine Transporters; Serotonin; Serotonin Transporters

Results 1-25 (49)