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.
serotonin (5-HT) release; 5-HT depletion; 5-HT transporter (SERT); SERT substrate; SERT-mediated current
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.
dopamine; transporter; amphetamine; Drosophila melanogaster; phosphatidylinositol 4,5-bisphosphate
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.
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.
Glycosylation; Heat Shock Protein (HSP); Heat Shock Protein 90 (Hsp90); Serotonin; Serotonin Transporter; Trafficking; Transporter
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.
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).
COMT; SLC6A4; BDNF; MRI; hippocampus; stress
•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”.
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
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.
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.
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
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.
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.
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.
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.
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.
addiction & Substance Abuse; blocker; catecholamines; designer drugs; neuropharmacology; Psychostimulants; releaser; transporter; designer drug; dopamine; cathinone; monoamine transporter; uptake blocker
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
Amphetamine; bath-salts; mass spectrometry; combo; psychostimulants; 2C-B; MDPV
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.
Bath salts; Cathinone; Designer drug; Dopamine; Mephedrone; Methylone; Monoamine transporter; Norepinephrine; Serotonin
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.
BAR domains; PDZ domains; protein–lipid interactions; receptors; transporters
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.
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.
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.
Dopamine Transporters; Endoplasmic Reticulum (ER); Membrane Transport; Monoamine Transporters; Serotonin; Serotonin Transporters