[Show abstract][Hide abstract] ABSTRACT: Neurotransmitter uptake 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 (N-) and carboxy (C-) terminal ends of the transporter. Although structures of homologs provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modelling, 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. 1-dimensional (1)H NMR and circular dichroism (CD) spectroscopy were consistent with 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 (FRET) signal between terminal CFP and YFP tags in full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well defined. Full-length models of SERT were generated that are consistent with these and published experimental data. The resultant models predict confined loci for the terminal domains whose separation increased during the transport-related conformational cycle, as predicted by structures of homologs and by the 'rocking bundle' hypothesis, and 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.
[Show abstract][Hide abstract] ABSTRACT: Addiction to psychostimulants (i.e., 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 behavioural 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.Neuropsychopharmacology accepted article preview online, 29 May 2014; doi:10.1038/npp.2014.124.
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 05/2014; · 8.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The A2A-receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein Gs occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A-adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C-terminus by examining diffusion of quantum dot labeled receptor variants in dissociated hippocampal neurons: single particle tracking of the A2A-receptor(1-311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was substantially reduced. A fragment comprising the SH3- and the guanylate kinase (GUK)-domain of synapse associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A-receptor C-terminus. Complex formation between the A2A-receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. Analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and thus promoted the accumulation of the A2A-receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A-receptor to a compartment with restricted mobility. In contrast, a mutated A2A-receptor (with (383)DVELL(387) replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A-receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.
Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. While there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different co-existing oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64, or via fusion to mGFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data is in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.
Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ibogaine is a psychoactive indole alkaloid. Its use as anti-addictive agent was accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by hERG potassium channel inhibition. Therefore, the interaction of ibogaine with hERG channels heterologously expressed in tsA-201 cells was studied in detail. Currents through hERG channels were blocked regardless whether ibogaine was applied via the extra- or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during the activation of the channels and was not observed for resting channels. With increasing depolarizations ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, while inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.
Journal of Pharmacology and Experimental Therapeutics 12/2013; · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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".
Neurochemistry International 11/2013; · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The A2A-adenosine receptor is a prototypical rhodopsin-like G protein-coupled receptor (GPCR), but has several unique structural features, in particular a long C-terminus (of > 120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A-receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A-receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem-affinity purification. The receptor/protein complexes were purified in quantities affording the analysis by mass spectrometry. We identified molecular chaperones (heat-shock protein HSP90α and HSP70-1A) that interact with and retain partially folded A2A-receptor prior to ER exit. Complex formation between the A2A-receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity-precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A-receptor. Finally, proteins of the HSP relay machinery (e.g., HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A-receptor. These observations are consistent with the proposed chaperone/coat protein complex-II (COPII) exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A-receptor. Release of HSP90 is required prior to recruitment of COPII components. This prevents premature ER export of partially folded receptors.
Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
Proceedings of the National Academy of Sciences 06/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the COPII component SEC24C for ER export. The closely related transporters for noradrenaline (NET) and dopamine (DAT) 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 ((607)RI(608) in SERT). Substituting K(610) by tyrosine, the corresponding residue found in NET and DAT, 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 (Y or V) in the +2 position specifies interaction with SEC24D, a hydrophilic residue (K, N, Q) 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.
Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.
Journal of Biological Chemistry 07/2012; 287(35):29627-35. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transporters for serotonin (SERT), dopamine, and noradrenaline have a conserved hydrophobic core but divergent N and C termini. The C terminus harbors the binding site for the coat protein complex II (COPII) cargo-binding protein SEC24. Here we explored which SEC24 isoform was required for export of SERT from the endoplasmic reticulum (ER). Three lines of evidence argue that SERT can only exit the ER by recruiting SEC24C: (i) Mass spectrometry showed that a peptide corresponding to the C terminus of SERT recruited SEC24C-containing COPII complexes from mouse brain lysates. (ii) Depletion of individual SEC24 isoforms by siRNAs revealed that SERT was trapped in the ER only if SEC24C was down-regulated, in both, cells that expressed SERT endogenously or after transfection. The combination of all siRNAs was not more effective than that directed against SEC24C. A SERT mutant in which the SEC24C-binding motif ((607)RI(608)) was replaced by alanine was insensitive to down-regulation of SEC24C levels. (iii) Overexpression of a SEC24C variant with a mutation in the candidate cargo-binding motif (SEC24C-D796V/D797N) but not of the corresponding mutant SEC24D-D733V/D734N reduced SERT surface levels. In contrast, noradrenaline and dopamine transporters and the more distantly related GABA transporter 1 relied on SEC24D for ER export. These observations demonstrate that closely related transporters are exclusive client cargo proteins for different SEC24 isoforms. The short promoter polymorphism results in reduced SERT cell surface levels and renders affected individuals more susceptible to depression. By inference, variations in the Sec24C gene may also affect SERT cell surface levels and thus be linked to mood disorders.
Journal of Biological Chemistry 03/2011; 286(18):16482-90. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.
Journal of Biological Chemistry 10/2010; 285(50):39201-10. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERT(T81A) and SERT(T81D), suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERT(T81A) in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERT(T81A) (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERT(T81A) were comparable with those through the wild type transporter. Both abundant Na(+) entry and accumulation of SERT(T81A) in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.
Journal of Biological Chemistry 04/2010; 285(14):10924-38. · 4.65 Impact Factor