InteractiveFly: GeneBrief

Serotonin transporter: Biological Overview | References

BIOLOGICAL OVERVIEW

The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. This study introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting (otherwise fully functional) hSERT-R⁶⁰⁷A on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-R⁵⁹⁹A led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. The possible physiological role of somatodendritic SERT was explored by comparing flies harboring wild type SERT and dSERT-R⁵⁹⁹A in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R⁵⁹⁹A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. It is concluded that in CSD neurons, (1) somatodendritic SERT supports ethanol attraction; (2) axonal SERT specifies ethanol aversion, and (3) the effect of axonal SERT can override that of somatodendritic SERT. These observations demonstrate a distinct biological role of somatodendritic and axonal serotonin transport (Kasture, 2019).

In the brain, the monoamines serotonin, noradrenaline and dopamine act as neuromodulators; monoaminergic axons originate from small clusters of neurons, elaborate complex arborizations and project diffusely to reach many neurons. This is exemplified by serotonergic projections, which originate from the midbrain and pontine raphe nuclei, and are found in virtually the entire mammalian brain. Serotonin released from these axonal projections is retrieved by the serotonin transporter (SERT) a member of the solute carrier-6 (SLC6) family (SLC6A4). SERT is the target for both, therapeutic and recreational drugs: inhibition of SERT by selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants (TCAs) is the basis for their eponymous action in depression and related mood disorders. Reverse transport and the resulting serotonin release underlie the action of methylene-dioxyamphetamine (MDMA/ecstasy) and related congeners (Sitte, 2015). SERT is not only found on the axolemma; circumstantial evidence suggested that it was also present on the cell bodies of raphe neurons. This was confirmed by electron microscopy: SERT was visualized on the somatodendritic surface, which raised the question of its physiological relevance (Kasture, 2019).

Axonal delivery of neurotransmitter transporters of the family SLC6 is contingent on export from the endoplasmic reticulum via the recruitment of the coatomer protein II (COPII) coat: the C-terminus of neurotransmitter transporter harbors an RL/RI-motif, which specifies their interaction with SEC24C or SEC24D, the cargo receptor of the COPII coat. Mutations in the RL/RI-motif of the transporter or in the cargo binding site of cognate SEC24 isoform results in somatodendritic confinement of the GABA-transporter- 1 (GAT1/SLC6A1) and of SERT. The physiological role of SERT located in the somatodendritic compartment is poorly understood, but it has been proposed to play an important role in the action of antidepressants (Kasture, 2019).

This addressed the physiological role of SERT in the somatodendritic compartment using the serotonergic system of Drosophila. The RI/RL-motif is conserved in most eukaryotic SLC6 transporters (Freissmuth, 2018) and present in the serotonin transporter of Drosophila melanogaster (dSERT). Flies also rely on orthologs of the COPII machinery, namely SEC23, SEC24AB, SEC24CD and SAR1 to support the ER export of natively folded membrane proteins (Kasture, 2017). The adult brain of Drosophila contains about 80 serotonergic neurons organized in distinct cell clusters, which are involved in modulating the circuitry underlying e.g. the circadian rhythm, feeding, aggression and long-term memory formation (Kasture, 2018). Furthermore, modulation of SERT expression in a subset of serotonergic neurons affects odor perception in adult flies (Xu, 2016). Accordingly, a point mutation was introduced in dSERT, and it was verified that the resulting mutant dSERT-R⁵⁹⁹A was enriched in the somatodendritic compartment of serotonergic neurons in the Drosophila brain. This experimental approach allowed for exploring the compartment-specific role of SERT in an in vivo context (Kasture, 2019).

Somatodendritic monoamine transporters presumably serve to clear the extracellular space of their cognate neurotransmitters and to replenish somatodendritic vesicles in a manner analogous to presynaptic transporters. However, their physiological role is much less understood than that of transporters residing in axonal arborizations of the monoaminergic neurons. It has been appreciated that somatodendritic release of dopamine, norepinephrine and serotonin occurs in the mammalian brain and that the underlying mechanisms differ from those regulating axonal release. In addition, somatodendritic DAT has been proposed to support dopamine release in the substantia nigra by switching into the reverse transport mode. It has, however, not been possible to examine, which biological responses, if any, depend on somatodendritic monoamine transporters. This study explored an approach to address this question by focusing on SERT, which is of relevance to mood disorders and pharmacotherapy. Three arguments support the conclusion that somatodendritic delivery of SERT is of physiological relevance. (1) The current experiments were designed to verify that a point mutation in the C-terminal motif, which is required for recruiting the cognate SEC24-isoform, i.e., mammalian SEC24C or Drosophila SEC24C/D, resulted in somatodendritic confinement of the resulting hSERT-R⁶⁰⁷A and dSERT⁵⁹⁹A, respectively: when expressed in primary raphe neurons, the hSERT-R⁶⁰⁷A stayed restricted in the somatodendritic compartment. Thus, the experiments hSERT-R⁶⁰⁷A recapitulated the findings with hSERT- RI^607,608AA (Montgomery, 2014) and ruled out that the inability to enter the axonal compartment was related to a folding defect. (2) The insights obtained from cultured neurons were confirmed in vivo: dSERT-R⁵⁹⁹A placed under the control of TRH-GAL4 was predominantly enriched in the somatodendritic compartment, while wild type dSERT was readily delivered to axonal territories delineated by the presynaptic marker synaptotagmin. This was independently verified by using a GAL4 driver line, which labels a single serotonergic neuron per hemisphere, namely the CSD neuron. Even if wild type dSERT and dSERT-R⁵⁹⁹A were co-expressed, they were subject to segregated delivery: wild type dSERT was prominently enriched in the antennal commissure, a defined axonal region, which was devoid of dSERT-R⁵⁹⁹A. These observations are consistent with earlier findings that the axonal delivery of SERT (Montgomery, 2014) and of GAT1 is contingent on COPII-dependent export from the ER. In fact, they rule out that this dependence arises from a cell culture artefact resulting from the absence of a trophic factor. Thus, dSERT- R⁵⁹⁹A is a valid experimental tool to address the role of somatodendritic SERT in vivo. (3) The CSD neurons play a modulatory role in the olfactory circuit. In the antennal lobes, CSD neurons forms synapses with the local interneurons and projection neurons to modulate the odor response. In a SERT-deficient background, the targeted reexpression of dSERT-R⁵⁹⁹A and of wild type dSERT in CSD neurons allowed for examining the relative importance of axonal and somatodendritic SERT in processing odorant cues. The results were unequivocal: somatodendritic dSERT-R⁵⁹⁹A sufficed to restore ethanol preference to levels seen in wild type flies. In contrast, the presence of wild type SERT in both, the axonal and somatodendritic compartment, produced ethanol aversion. Flies harboring dSERT-R⁵⁹⁹A differ from those expressing wild type SERT by the absence of axonal transport activity. Because the presence of dSERT-R⁵⁹⁹A in CSD neurons sufficed to restore ethanol preference, it is safe to conclude that somatodendritic SERT supports ethanol attraction. SERT in the axonal compartment of CSD neurons specifies ethanol aversion. In the processing of olfactory cue, the effect of axonal SERT overrides the action of somatodendritic SERT. Thus, these findings demonstrate a distinct biological role of somatodendritic and axonal serotonin transport (Kasture, 2019).

Expression of transgenes placed under the control of TRH-GAL4 is achieved in the vast majority (75%-100%) of serotonergic neurons (Alekseyenko, 2010). The dichotomy of wild type dSERT and somatodendritically confined dSERT-R⁵⁹⁹A was recapitulated, if their expression was driven by TRH-GAL4: ethanol preference was restored by reexpression of wild type SERT but not of dSERT-R⁵⁹⁹A. For both, wild type SERT and dSERT-R⁵⁹⁹A, the odorant response differed after CSD-restricted and TRH-GAL4-mediated reexpression. This apparent discrepancy can be rationalized by taking into account that two additional clusters of serotonergic neurons in IP and LP1 impinge on the circuitry, which processes olfactory cues. Stimulation of IP and LP1 antagonize the action of CSD neurons (Xu, 2016). While the precise hierarchy of the three serotonergic neurons in IP, LP1 and CSD is not known, it is clear that modulation of the ethanol preference depends on the activity of SERT: administration of the SSRI citalopram eliminated all changes in ethanol preference induced by forced expression of wild type dSERT or of dSERT-R⁵⁹⁹A, regardless of whether global reexpression or CSD-restricted expression was examined. Thus, the current observations support the conclusion that axonal rather than somatodendritic serotonin transport in IP and LP1 serotonergic neurons is required to maintain ethanol preference (Kasture, 2019).

Variations in SERT levels are thought to have an impact on the susceptibility of people for depression and anxiety: this is exemplified by a polymorphism in the promoter, where the short allele, which results in lower expression levels, is associated with a higher propensity of people to develop depression and anxiety. It is not clear to which extent this increased susceptibility is affected by the levels of presynaptic and somatodendritic SERT. A bioinformatics study mined databases for possible disease markers, which were relevant for psychiatric disorders, i.e., schizophrenia, major depression and bipolar disease: microarray data were collected from Brodmann area 10 of the prefrontal cortex of the patients. Genes, which differed from those of samples from control patients, were mapped onto protein-protein interaction networks extracted from several databases. This exercise showed that patients suffering from bipolar disorder have altered SEC24C levels. In fact, SEC24C was ranked first among 104 altered genes based on the error of probability. It is worth noting that SERT could not be present in these transcripts: in the adult human brain, SERT is synthesized in the somata of the serotoninergic neurons of the raphe nuclei. Hence, the mRNA encoding SERT is not present in Brodmann area -10. Accordingly, SERT was not represented in the protein-protein interaction networks mapped in another study. The current findings highlight the importance of COPII dependent export of SERT from the ER in specifying axonal delivery of SERT in vivo. Loss of SEC24C is incompatible with normal brain development, because SeC24C-deficient neurons are prone to apoptosis. However, the gene encoding SEC24C is highly polymorphic with e.g., 12 non-synonymous variants in the coding sequence. It is therefore plausible that variations in SEC24C have an impact on the relative levels of SERT in the axonal and the somatodendritic compartment. This may be of relevance in defining the susceptibility to mood disorders. The current observations show that in flies axonal and somatodendritic serotonin transport support distinct behavioral responses. Some expression of dSERT-R⁵⁹⁹A was also seen in the fan-shaped body, which (based on synaptotagmin) is an axonal territory of serotonergic neurons. This suggests that in some neurons, a fraction of dSERT-R⁵⁹⁹A can enter the axonal compartment. This is reminiscent of GAT1, where the corresponding mutant was also delivered at low levels to the axons. The underlying mechanisms are not clear: possibly high levels of SEC24CD can overcome the effect of the point mutation in the SEC24-binding motif. This limitation does not detract from the major difference in the localization of wild type dSERT and dSERT-R⁵⁹⁹A and from the major difference in their effect on the processing of odorant cues. In depression, there is a delay in the onset of the therapeutic response to SSRIs and tricyclic antidepressants. A widely accepted model posits that inhibition of SERT leads to progressive desensitization of 5HT1A-receptors, which act as autoreceptors in the dorsal raphe nuclei. The resulting decline in 5HT1A-autoreceptor activity relieves the inhibition imposed by serotonin and thus allows for gradual recovery of the serotonergic neurons in the dorsal raphe such that their activity is restored. This hypothetical sequence of events accounts for the delayed onset of the antidepressant action. It is not clear, if inhibition of SERT takes place on the presynaptic side, i.e., on recurrent collaterals of the dorsal raphe, or on the somatodendritic side. In fact, the number of serotonergic fibers in the dorsal raphe is low. Hence, it is conceivable that inhibition of somatodendritic SERT is more important to explain the antidepressant action than that of axonal SERT. The availability of a SERT variant, which is enriched in the somatodendritic compartment, allows for exploring this conjecture. It may also shed light on the role of SERT distribution in the development of mood disorders (Kasture, 2019).

Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in 'simpler' animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. This study has advanced understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. In doing this, a mutant for Drosophila SERT (dSERT) was characterized, and additionally a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), was used, whose mechanism of action involves the SERT protein. The results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior, and decreased levels in an anxiety-related parameter, centrophobism. It was also shown that 4-MTA increases the negative chemotaxis toward a strong aversive odorant, benzaldehyde. The neurochemical data suggest that this effect is mediated by dSERT and depends on the 4-MTA-increased release of serotonin in the fly brain. In silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum, this neurochemical, in silico, and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila (Hidalgo, 2017).

A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila

Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. This study dissected the function of the key regulator of serotonin signaling-the serotonin transporter-in innate olfactory attraction to ethanol in Drosophila melanogaster. A mutated version of the serotonin transporter was generated that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. Four serotonergic neurons were identified that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. These results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior (Xu, 2016).

Olfactory attraction to ethanol is regulated by two sets of serotonergic neurons. Two neurons -- the serotonergic CSD neurons -- counteract the inhibitory function of a second set of four serotonergic neurons. The enhancement of sensory input by altered serotonin signaling of the CSD neurons is stronger than more internal information that might influence the behavioral outcome. The observation that strong sensory information overrules internal conditions unravels a hierarchy of information that governs behavioral output (Xu, 2016).

Prolonged serotonin signaling in approximately 83% of the serotonergic neurons using the Trh-Gal4 driver results in normal degree of attraction to 5% EtOH containing food odors. At the first glance, these finding suggest that dSert in these broad set of serotonergic neurons is not involved in the regulation of ethanol attraction. This apparently normal behavior is shared by other Sert knock out animals. For example Sert knock out mice develop normal conditioned place preference -- a model for the cocaine reward -- despite the fact that cocaine binds to Sert. However, breaking down the serotonergic networks underlying the regulation of the attraction to ethanol revealed that a dysregulation of serotonergic neurons can phenocopy normal behavior and that the identified serotonergic neurons under normal circumstance indeed regulate the olfactory attraction. The results show that compensation can occur on network level. The serotonergic neurons underlying the gating of olfactory attraction to ethanol innervate distinct brain regions normally involved in the regulation of olfactory responses. The attraction promoting serotonergic CSD neuron receives synaptic input from the ipsilateral antennal lobes from a broad set of glomeruli, collecting and exchanging olfactory information with the mushroom bodies and lateral horn and releasing information to the contralateral antennal lobe regions (Xu, 2016).

The second set of inhibitory serotonergic neurons includes one neuron each from the IP and LP1 clusters that suppresses innate attraction to ethanol odor. The neuron of the IP cluster projects into the superior medial protocerebrum (smpr), posterior lateral protocerebrum (plpr), and the optic system. The smpr receives olfactory output both directly from the antenna lobes and indirectly from lobes of the LH. The LP1 neuron innervates the ventral lateral protocerebrum (vlpr), a region implicated as a multimodal integration center of sensory stimuli including olfactory information. This multimodal nature of this brain region is further supported by recent finding that a pair of similar serotonergic neurons is involved in aggression and mediating the hunger state of the fly. The fact that the alteration of serotonin signaling in the CSD neuron bypasses the inhibitory effect of the Sert3-Gal4-dependent neurons further supports that the inhibitory effect of those neurons is not due to loss of olfactory perception. Combined with the observation that activation of the Sert3-Gal4-dependent neurons alone does not cause attraction or aversion, these results show that these neurons regulate the execution of a function rather than being involved in the initial step of odor information processing or execution of the approach behavior. This is consistent with a role for prolonged serotonin signaling in response inhibition. This suppression of innate olfactory attraction to ethanol can be overruled by strengthening the constant sensory input to the olfactory system. The consequence of the failing inhibitory mechanism is that the likelihood of exposure to food odors containing ethanol increases, and the opportunity to ingest ethanol might increase in turn (Xu, 2016).

The observation that strong sensory information overrules internal conditions also suggests that under normal circumstances both mechanisms require a tight temporal control. That timing matters is further substantiated by the observations that short term systemic application of a serotonin precursor and short term activation of the Sert3-Gal4 dependent subset of serotonergic neurons using opto-genetics reduce ethanol attraction, but long term intervention of serotonin signaling by dSertDN expression under the control of the Trh-Gal4 driver did not interfere with ethanol attraction. However the regulation of innate ethanol odor attraction is even more complex since other neurotransmitter systems are also involved in regulating the approach to an olfactory stimulus. Previous work has shownn that olfactory ethanol attraction depends on the reinforcement of the olfactory information by a subset of octopaminergic/tyraminergic neurons and the aversion depends on the activation of a subset of dopaminergic neurons acting as a negative reinforcer (Schneider, 2012). This study shows that the maintenance of olfactory information by the serotonergic CSD neurons promote the approach to the ethanol food odor mixture. In the presence of prolonged external olfactory information, the inhibitory information of an internal serotonergic reference system is overruled. The regulation of behavioral outcome requires a tight temporal regulation between the positive and negative reinforcing, inhibiting and prolonging mechanisms depending on the location of the fly relative to the ethanol containing food source and its internal condition. It will be interesting to see where the four mechanisms intersect with each other, how they are orchestrated and whether they are sufficient to regulate the attraction to ethanol. Furthermore, given the evolutionarily conserved nature of the serotonin transporter and the wide expression of serotonin, the serotonergic gating mechanism might delineate a basic principle for other behaviors and organisms as well (Xu, 2016).

Mechanism of paroxetine (Paxil) inhibition of the serotonin transporter

The serotonin transporter (SERT) is an integral membrane protein that exploits preexisting sodium-, chloride-, and potassium ion gradients to catalyze the thermodynamically unfavorable movement of synaptic serotonin into the presynaptic neuron. SERT has garnered significant clinical attention partly because it is the target of multiple psychoactive agents, including the antidepressant paroxetine (Paxil), the most potent selective serotonin reuptake inhibitor known. However, the binding site and orientation of paroxetine in SERT remain controversial. To provide molecular insight, this study constructed SERT homology models based on the Drosophila melanogaster dopamine transporter, and paroxetine was docked to these models. The predicted binding configurations were tested with a combination of radioligand binding and flux assays on wild-type and mutant SERTs. The data suggest that the orientation of paroxetine, specifically its fluorophenyl ring, in SERT's substrate binding site directly depends on this pocket's charge distribution, and thereby provide an avenue toward understanding and enhancing high-affinity antidepressant activity (Davis, 2016).

Paroxetine is the most potent SERT inhibitor and one of the most effective therapeutics currently available for a broad spectrum of neuropsychiatric illnesses, yet its precise molecular interactions within its binding site have remained elusive. Part of this dearth of knowledge stems from the paucity of studies expressly targeted toward paroxetine, unlike the prototypical antidepressants escitalopram or imipramine, but much of the deficiency is simply due to the unfortunate outcome of not having identified an amino acid which, when mutated, influences the affinity of paroxetine as much as it does that of other antidepressants. One conceivable exception is a report that characterized a series of cross-species chimeras between ggSERT and hsSERT followed by selected site-directed mutants and conjectured that positions 169 as well as 172 in hsSERT play important roles in 'sensing the N-methylation state of SERT antagonists' (Larsen, 2004; Davis, 2016).

Prior investigations into the delineation of antagonist binding sites in SERT have relied on both cross-species comparisons or, since the arrival of the first LeuT structures, docking of drugs into reliable homology models. This study has integrated the strengths of the two techniques to interrogate the atomic origins of paroxetine's specificity and extraordinarily high affinity (Davis, 2016).

A peculiar property of the three SERT homologues that was employed in this work is the lack of a direct correlation between paroxetine potency and the degree of sequence identity. Despite the fact that hsSERT shares 82% identity with ggSERT (chicken) compared with 52% for dmSERT, the paroxetine potency of ggSERT is at least 10-fold lower. This apparent paradox can be reconciled by the data which show that dmSERT possesses at least two 'compensatory' residues at positions P488 (T497 in hsSERT) and I492 (V501 in hsSERT) that appear to offset the low identity. For example, introduction of the corresponding hsSERT residue at these positions in dmSERT (P488T and I492V) led to diminished potencies relative to that for dmSERT-WT. These compensatory amino acids apparently create an environment in which a single-residue exchange at position 164 (169 in hsSERT) completely switches potencies between the hsSERT and dmSERT homologues. In fact, as mentioned above, this single-residue exchange actually improves dmSERT's paroxetine potency 2.3-fold beyond that of hsSERT and reduces hsSERT's paroxetine potency 2.4-fold below that of dmSERT. The docking results indicate that positioning of the benzodioxol group in paroxetine may not be the same in the two proteins, reflecting the differences in protein environment within subsite C. Nevertheless, the presence of the aspartate or alanine at position 169 leads to 7-fold reciprocal changes in potency and this correlates directly with the position of the fluorophenyl group. Compared with hsSERT-WT, the fluorophenyl moiety of paroxetine is predicted to bind to hsSERT-A169D in a less deeply buried orientation. Note that the deeper insertion is also not predicted for ggSERT or dmSERT, both of which have an aspartate at the equivalent position. Interestingly, while the apparent affinity of ggSERT-D209A for paroxetine improves relative to that of ggSERT-WT, it still falls ~5-fold short relative to that of hsSERT-WT. It is speculated that the effect of the I172/V212 substitution is additive to that of the A169/D209 replacement potentially accounting for the vestigial change in potency between ggSERT-D209A and hsSERT-WT (Davis, 2016).

In summary, by employing three, rather than only two, SERT homologues, in combination with computational biology and functional analyses, this study has not only confirmed the importance of the previously-identified position 169 and, to a lesser extent, 172, but are also the first to implicate electrostatic contributions in the recognition between a specific functional group of the SSRI paroxetine and a specific SERT position. Although a full SAR study will be required to comprehensively dissect other elements of paroxetine selectivity and especially potency, which is beyond the scope of this work, this study has unveiled a pivotal factor in paroxetine-SERT interactions that can now serve as a launching point for future strategic drug development with piperidine derivatives (Davis, 2016).

The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for MDMA translocation and the induction of substrate efflux

The substituted amphetamine MDMA is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic. Previous, studies has identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. This study performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. The findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser (Sealover, 2016).

The serotonin transporter expression in Drosophila melanogaster

The serotonin transporter is an important regulator of serotonergic signaling. In order to analyze where the Drosophila melanogaster ortholog of the mammalian serotonin transporter (dSERT) is expressed in the nervous system, a dSERT antibody serum was used. Ectopic expression studies and loss of function analysis revealed that the dSERT antibody serum specifically recognizes dSERT. It was shown that in the embryonic nervous system dSERT is expressed in a subset of Engrailed-positive neurons. In the larval brain, dSERT is exclusively expressed in serotonergic neurons, all of which express dSERT. dSERT-positive neurons surround almost all brain neuropiles. In the mushroom body of the adult brain, extrinsic serotonergic neurons expressing dSERT engulf the mushroom body lobes. These neurons show regional differences in dSERT and serotonin expression. At the presynaptic terminals, serotonin release is sterically linked to serotonin reuptake. In contrast to this, there are other areas in serotonergic neurons where dSERT expression and/or function are uncoupled from synaptic neurotransmitter recycling and serotonin release. The localization pattern of dSERT can be employed to further understanding and analysis of serotonergic networks (Giang, 2011).

Serotonergic dystrophy induced by excess serotonin

Administration of certain serotonin-releasing amphetamine derivatives (fenfluramine and/or 3,4-methylenedioxymethamphetamine, MDMA, 'ecstasy') results in dystrophic serotonergic morphology in the mammalian brain. In addition to drug administration, dystrophic serotonergic neurites are also associated with neurodegenerative disorders. This study demonstrates that endogenously elevated serotonin in the Drosophila CNS induces aberrant enlarged varicosities, or spheroids, that are morphologically similar to dystrophic mammalian serotonergic fibers. In Drosophila these spheroids are specific to serotonergic neurons, distinct from typical varicosities, and form only after prolonged increases in cytoplasmic serotonin. These results also suggest that serotonin levels during early development determine later sensitivity of spheroid formation to manipulations of the serotonin transporter (SERT). Elevated serotonin also interacts with canonical protein aggregation and autophagic pathways to form spheroids. The data presented in this study support a model in which excess cytoplasmic neurotransmitter triggers a cell-specific pathway inducing aberrant morphology in fly serotonergic neurons that may be shared in certain mammalian pathologies (Daubert, 2010).

Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter [LeuT(Aa)] structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables critical assessment and validation og their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. These studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected (Kaufmann, 2009).

Conserved serine residues in serotonin transporter contribute to high-affinity cocaine binding

Serotonin transporter (SERT) is one of the key protein targets of cocaine. Despite intensive studies, it is not clear where cocaine binds to its targets and what residues are involved in cocaine binding. This study cloned the serotonin transporter from silkworm (Bombyx mori, bmSERT). When expressed in cultured cells, bmSERT is over 20-fold less sensitive to cocaine than Drosophila melanogaster SERT (dmSERT). Species-scanning mutagenesis was performed using bmSERT and dmSERT. There are two adjacent threonine residues in transmembrane domain 12 of bmSERT where the corresponding residues are two serines in dmSERT and in all known mammalian monoamine transporters. Replacing the serine residues with threonines in dmSERT reduces cocaine sensitivity; while switching the two threonine residues in bmSERT to serines increased cocaine sensitivity. Mutations at the corresponding residues in dopamine transporter also changed cocaine affinity. These results suggest that the conserved serine residues in SERT contribute to high-affinity cocaine binding (Gu, 2006).

Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT

The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. This study examined the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. A surprising limitation was found on 5-HT storage following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT (Park, 2006).

Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT

The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. This study examine the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. A surprising limitation on 5-HT storage was found following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT (Park, 2006).

The simplest explanation for this inability to detect intracellular 5-HT is that there is a cell-type-specific limitation on dSERT function. Arguing for this interpretation is the finding that most if not all neurons will nonspecifically take up 5-HT if incubated at a sufficiently high concentration of 5-HT (10 mM), that lack of expression of the vesicular monoamine transporter dVMAT does not affect the storage ability of neurons, and that no increased levels of the expected 5-HT metabolite N-Ac-5-HT is detected in CNS with ectopic dSERT expression. Part but not all of the limitation is due to limited intracellular diffusion of 5-HT, because incubation in a more moderate concentration of 5-HT, 1 mM, leads to a moderate expansion in the number of cells that can uptake 5-HT, but in a dSERT-dependent manner (Park, 2006).

Nevertheless, this interpretation would stand in contrast to conventional wisdom regarding vertebrate amine transporters. Vertebrate amine transporters can be readily expressed in several types of non-neural transformed cell lines as well as in Xenopus oocytes, with only small differences in their functional properties as a function of cellular host, leading to the general view that the nature of the host cell is not critical. Because dSERT can also be expressed in heterologous cell types including Drosophila S2 cells, it seems that the critical difference between this study versus previous studies is the site of expression, the nerve cord of living flies. There are no analogous amine transporter overexpression studies in the vertebrate nervous system, and it is possible that similar mechanisms might act to limit expression of these transporters to appropriate cell types (Park, 2006).

There is much evidence indicating that amine transmitter transporters are subject to post-translational modifications, including glycosylation and phosphorylation. In particular, roles for protein kinase C (PKC) and phosphatase 2A (PP2A) have become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter internalization and loss of functional uptake capacity. In addition, p38 MAPK activation can stimulate hSERT activity in CHO cells without affecting trafficking to the cell surface (Park, 2006).

Multiprotein complexes that include the transporters, scaffolding proteins, kinases, and phosphatases also can affect their activity and propensity to undergo membrane internalization. Any of these proteins could be candidates for a factor limiting dSERT function to specialized types of neurons in the fly CNS. The only limitation on this factor(s) is that it would not appear to limit the membrane appearance of dSERT, that is, that it does not appear to function at an early step in dSERT Golgi maturation, because GFP-linked dSERT shows similar membrane association at the level of light microscopy in all cell types. In rodents, transient expression of SERT and VMAT2 allows the storage and release of 5-HT in developing thalamocortical projections, despite the absence of serotonin biosynthesis in these cells. It has been assumed that VMAT2 and SERT expression are sufficient to allow uptake of 5-HT synthesized at exogenous sites, and storage in the thalamic neurons. It is possible that the additional 5-HT uptake/accumulation that that was observed following incubation in 1 mM 5-HT in the presumptive adult neuroblasts and their progeny reflects a similar phenomenon, transient expression of dSERT in a subset of developing neurons. More importantly, however, the vast majority of neurons do not accumulate 5-HT even in the presence of exogenous 5-HT. Thus, these data suggest that regulated expression of additional factors may be needed to accomplish a change in neurochemical identity. Cell-type-specific ectopic expression of dSERT in Drosophila will be a favorable tool by which to search genetically for factor(s) limiting 5-HT storage activity (Park, 2006).

robo2 and robo3 interact with eagle to regulate serotonergic neuron differentiation

The function of the central nervous system (CNS) depends crucially upon the correct differentiation of neurons and formation of axonal connections. Some aspects of neuronal differentiation are known to occur as axonal connections are forming. Although serotonin is a highly conserved neurotransmitter that is important for many CNS functions, little is known about the process of serotonergic neuron differentiation. In Drosophila, expression of the serotonin transporter (SerT) is both temporally and physically related to midline crossing. Additionally, the axon guidance molecules roundabout2 and roundabout3 (robo2/3) are necessary for serotonergic neuron differentiation and function independently of their ligand, slit. Loss of robo2 or robo3 causes a loss of SerT expression in about half of neurons, and resembles the phenotype seen in mutants for the transcription factor eagle (eg). A direct relationship is shown between robo2/3 and eg: robo2/3 mutants lose Eg expression in serotonergic neurons, and robo2 and eg interact genetically to regulate SerT expression. It is proposed that post-midline expression of Robo2/3 is part of a signal that regulates serotonergic neuron differentiation and is transduced by the transcription factor Eg (Couch, 2004).

Thus SerT expression in the fly is temporally and physically tied to axon guidance across the midline. The data further indicate that the axon guidance molecules robo2 and robo3 (robo2/3) positively regulate serotonergic neuron differentiation; a loss of robo2/3 function causes a loss of SerT expression in ~50% of neurons. A robo2/3 loss of function closely resembles an eg mutant phenotype. Finally, the data show a dose-sensitive relationship between Robo2/3, Eg and SerT expression, suggesting that they function in the same genetic pathway to control serotonergic neuron differentiation. This interpretation is supported by the fact that loss of robo2 or robo3 causes a loss of Eg expression, and by genetic rescue experiments (Couch, 2004).

By visualizing serotonergic axonal projections with tau-lacZ, it was determined that SerT expression begins at the end of stage 15, just after growth cones complete midline crossing and reach the contralateral side. This temporal correlation between midline crossing and SerT induction suggests that the midline is important for serotonergic neuron differentiation in the fly, as it is in the grasshopper (Condron, 1999). Further evidence for the importance of the midline comes from data showing that in wild-type cords, axons physically separated from the midline fail to express SerT. These results recapitulate similar experiments in the grasshopper. Additionally, when the repulsive axon guidance receptor Unc5 is expressed in serotonergic neurons, a partial loss of SerT expression is observed. Although these results suggest a role for the midline in serotonergic neuron differentiation, it remains unclear whether this role is temporally restricted as it is in the grasshopper, and, additionally, what factors act as the presumptive midline signal. FGF signaling in the grasshopper is crucial for SerT induction (Condron, 1999), and plays a role in the differentiation of vertebrate serotonergic neurons. In the fly, experiments indicate that FGF signaling also appears to be important for SerT regulation (Couch, 2004).

One problem with interpreting the role for the midline is the lack of an abnormal serotonergic phenotype in mutants for the master regulatory gene sim, where midline cells fail to properly differentiate. It is difficult to speculate about what factors may allow normal differentiation in the absence of normal midline cells, since there are many changes in gene regulation throughout sim mutants. Although the results suggest a role for the midline in serotonergic neuron differentiation, it is likely to be more complicated than a simple switch acting to induce differentiation (Couch, 2004).

The data show that a loss of robo2 or robo3 causes a loss of SerT expression, suggesting that Robo2/3 function positively to regulate serotonergic neuron differentiation. A positive role for Robo2 is further supported by results showing that overexpression of Robo2 prevents a loss of SerT in neurons physically separated from the midline. Possibly, Robo2 functions downstream of the midline signal required for SerT induction and thus allows differentiation to proceed in the absence of such a signal. An alternative hypothesis suggests that Robo2/3 function indirectly to induce SerT, by guiding serotonergic axons to an unknown signal in the contralateral neuropil. Such indirect signaling occurs in the developing vertebrate CNS where trophic support is required by commissural axons at the floorplate, an intermediate axonal target. Although the possibility that Robo2/3 act indirectly to regulate serotonergic neuron differentiation cannot be ruled out at this time, several lines of evidence suggest a more direct role. These data shows that overexpression of Robo2 not only spares SerT loss following a midline cut but also rescues an eg hypomorph, and furthermore, that an Eg gain of function rescues a robo2 loss of function; these results strongly suggests that Robo2 functions autonomously in the serotonergic neurons. Additionally, SerT loss is not seen in other guidance mutants that disrupt midline crossing or cause general disorganization of the CNS. However, it is difficult to clearly resolve the presence of Robo2/3 protein specifically in the serotonergic neurons because of the broad distribution of neuronal processes and the fact that serotonergic neuron branching does not correspond simply to any Fas2 pathway where axons are known to express Robo2/3 (Couch, 2004).

Distinct molecular recognition of psychostimulants by human and Drosophila serotonin transporters

In this study, human embryonic kidney (HEK)-293 cells stably expressing human, Drosophila, or a chimeric serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, dSERT, and H(1-281)D(282-476)H(477-638), respectively) were used to explore the ability of two libraries of structurally distinct psychostimulants to inhibit 5-HT uptake. One library consisted of 3-phenyltropane analogs, whereas the second library consisted of several substituted amphetamines. hSERT exhibited a lower K(i) value for all the compounds in both libraries compared with dSERT, whereas the chimeric SERT exhibited properties more closely resembling those of dSERT. This species selectivity was explored using computer-generated comparative molecular field analysis to model the interactions of the cocaine analogs and substituted amphetamines at hSERT, dSERT, and the cross-species chimera. Models for the 3-phenyltropane analogs indicate that a region exists around the aromatic ring where decreased electron density is favored, particularly for hSERT. This finding may indicate pi-pi stacking with an aromatic amino acid residue in SERT. Also, electronegative substituents in the 4'-position provide favorable interactions. This structural feature was demonstrated by increased potency of analogs with electronegative substituents on the aromatic ring that withdraw electron density. For the substituted amphetamines, key areas for interaction exist around the amine, an electrostatic component surrounding the 3-position on the aromatic ring, and a steric component surrounding the 4-position (Roman, 2004).

Distinct recognition of substrates by the human and Drosophila serotonin transporters

The human and Drosophila serotonin transporters (hSERT and dSERT, respectively) were used to explore differences in substrate properties. hSERT and dSERT showed similar Km values for 5-hydroxytryptamine (5-HT; serotonin) transport (1.2 and 0.9 micro M, respectively), suggesting similar recognition of 5-HT by the two species variants. Although dSERT cell surface expression was approximately 8-fold lower than that of hSERT, dSERT does appear to have a 2-fold faster turnover number for inward transport of 5-HT. Interestingly, another substrate, N-methyl-4-phenylpyridinium (MPP+), was transported only by hSERT. However, MPP+ inhibited 5-HT uptake in both species variants with similar potencies. Two cross-species chimeras, H1-118D119-627 and H1-281D282-476H477-638, were also unable to transport MPP+, implicating the role of transmembrane domains V to IX in the substrate permeation pathway. Based on exchange experiments, certain substituted-amphetamines also appear to be poor substrates at dSERT. Two-electrode voltage-clamp studies in oocytes confirmed that the amphetamines do not possess substrate-like properties for dSERT. The data suggest distinct molecular recognition among SERT substrate classes that influence translocation mechanisms (Rodriguez, 2003).

Substrates and temperature differentiate ion flux from serotonin flux in a serotonin transporter

Neurotransmitter transporters couple the transport of transmitter against its concentration gradient to the electrochemical potential of associated ions which are also transported. Recent studies of some neurotransmitter transporters show them to have properties of both traditional carriers and substrate-dependent ion channels, in that ion fluxes are in excess of that predicted from stoichiometric substrate fluxes. Whether these properties are comparable for all transporters, the extent to which these permeation states are independent, and whether the relationship between these two states can be regulated are not well understood. To address these questions, this study expressed the Drosophila serotonin (5HT) transporter (dSERT) in Xenopus oocytes and measured both substrate-elicited ion flux and 5HT flux at various temperatures and substrate concentrations. The ion flux and 5HT flux components of the transport process have a significant temperature dependence suggesting that ion flux and transmitter flux were found to arise from a similar thermodynamically-coupled process involving large conformational changes (e.g., gating). These data are in contrast to those shown for glutamate transporters, suggesting a different permeation process for 5HT transporters. The relationship between ion flux and 5HT flux is differentially regulated by chloride and 5HT, suggesting that these permeation states are distinct. The difference in half-maximal 5HT concentration necessary to mediate ion flux and 5HT flux occurs at submicromolar 5HT concentrations suggesting that the relative participation of dSERT in ion flux and 5HT flux will be determined by the synaptic 5HT concentration (Beckman, 2001).

Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is responsible for the inactivation of synaptic 5-HT and is also a target for multiple psychostimulants. Despite the critical role of SERT in 5-HT inactivation and psychostimulant response, many aspects of the transporter's recognition of ligands are poorly defined. This study took advantage of sequence divergence of SERT species variants to identify structural determinants of substrate recognition. Tryptamine derivatives with substitutions at the 4 and 7 positions on the phenyl ring, the indole nitrogen, and the beta position show up to 40-fold potency differences for inhibiting [(3)H]5-HT transport in cells transfected with either human or Drosophila melanogaster SERT cDNAs. Species selectivities of these derivatives were largely recapitulated in antagonist binding. Human/D. melanogaster SERT chimera studies implicated the first two SERT transmembrane domains (TMDs) in the potency of the indole nitrogen-substituted compounds N-isopropyltryptamine (NIT), 5-methoxy-N-isopropyltryptamine (5-MNIT), and the 7-substituted compound 7-benzyloxytryptamine (7BT). Potency differences of analogs with substitutions at the 4 and beta positions are influenced by sequences distal to this region. Within TMD I-II, species-scanning mutagenesis implicated a single residue (Y95 in human SERT, F90 in D. melanogaster SERT) in the recognition of NIT, 5-MNIT, and 7BT. Remarkably, this is the same site that was established previously in species-specific recognition of the antagonists citalopram and mazindol. These findings support a critical role for TMD I residues in defining shared aspects of SERT substrate and antagonist recognition (Adkins, 2001).

Ionic interactions in the Drosophila serotonin transporter identify it as a serotonin channel

Serotonin transporters (SERTs) are targets for drugs such as Prozac that increase serotonin (5HT) levels by blocking 5HT reuptake. Although SERTs saturate in the micromolar range, synaptic 5HT may exceed 1 mM. To examine SERT's response to high 5HT concentrations, Drosophila SERT (dSERT) was expressed in Xenopus oocytes; transport continued to increase with concentration up to 0.3 mM 5HT. As 5HT is a monovalent cation, its entry through an ion channel in SERT might explain uptake at high concentrations. This study therefore investigated dSERT using traditional ion channel methods, including mole-fraction experiments under voltage clamp. It is proposed that SERTs may function as 5HT-permeable channels, and that this mechanism may be important for clearance of the neurotransmitter at high concentrations (Petersen, 1999).

Drosophila serotonin transporters have voltage-dependent uptake coupled to a serotonin-gated ion channel

Serotonin (5HT) transporters (SERTs) couple to existing ion gradients to transport 5HT into presynaptic terminals. In mammalian SERTs, the transport cycle is reported as electroneutral, with a translocation of zero net charge, and 5HT uptake is independent of membrane voltage. Yet mammalian SERTs exhibit 5HT-induced currents, and Drosophila SERTs (dSERTs) show voltage-dependent uptake. Thus, the relationship between uptake and current remains controversial; furthermore, the number of 5HT molecules translocated per ion channel event is unknown. To investigate this, heterologous expression of cloned dSERTs was used to measure 5HT flux and dSERT currents concurrently under voltage clamp, and fluctuation analysis was used to measure the size of the elementary ionic events in the same cells. RNA-injected Xenopus oocytes accumulate 5HT, and paroxetine or desipramine inhibit this uptake. RNA-injected oocytes also display paroxetine-sensitive 5HT-induced currents and 5HT-independent leak currents. Na replacement decreases the uptake and the induced currents. 5HT-induced current and 5HT uptake both increase at negative potentials, where 5HT carries approximately 5% of the induced current. Recently, several groups have reported similar phenomena for other transporters, in which transmitter-induced currents exceed the predictions of coupled transport. This study provides evidence that in dSERT, approximately 500 5HT molecules are translocated per channel opening, which, at -20 mV, carries approximately 10,000 electronic charges. These data support a model in which 500 SERT cycles occur for each 5HT-induced channel opening or a model in which 500 5HT molecules and 10,000 electronic charges pass through a common pore (Galli, 1997).

Dorsal raphe dual serotonin-glutamate neurons drive reward by establishing excitatory synapses on VTA mesoaccumbens dopamine neurons

Dorsal raphe (DR) serotonin neurons provide a major input to the ventral tegmental area (VTA). This study shows that DR serotonin transporter (SERT) neurons establish both asymmetric and symmetric synapses on VTA dopamine neurons, but most of these synapses are asymmetric. Moreover, the DR-SERT terminals making asymmetric synapses on VTA dopamine neurons coexpress vesicular glutamate transporter 3 (VGluT3; transporter for accumulation of glutamate for its synaptic release), suggesting the excitatory nature of these synapses. VTA photoactivation of DR-SERT fibers promotes conditioned place preference, elicits excitatory currents on mesoaccumbens dopamine neurons, increases their firing, and evokes dopamine release in nucleus accumbens. These effects are blocked by VTA inactivation of glutamate and serotonin receptors, supporting the idea of glutamate release in VTA from dual DR SERT-VGluT3 inputs. These findings suggest a path-specific input from DR serotonergic neurons to VTA that promotes reward by the release of glutamate and activation of mesoaccumbens dopamine neurons (Wang, 2019).

Structural basis for recognition of diverse antidepressants by the human serotonin transporter

Selective serotonin reuptake inhibitors are clinically prescribed antidepressants that act by increasing the local concentrations of neurotransmitters at synapses and in extracellular spaces via blockade of the serotonin transporter. This study report X-ray structures of engineered thermostable variants of the human serotonin transporter bound to the antidepressants sertraline, fluvoxamine, and paroxetine. The drugs prevent serotonin binding by occupying the central substrate-binding site and stabilizing the transporter in an outward-open conformation. These structures explain how residues within the central site orchestrate binding of chemically diverse inhibitors and mediate transporter drug selectivity (Coleman, 2018).

Serotonin transporter associated protein complexes are enriched in synaptic vesicle proteins and proteins involved in energy metabolism and ion homeostasis

The serotonin transporter (SERT) mediates Na(+)-dependent high-affinity serotonin uptake and plays a key role in regulating extracellular serotonin concentration in the brain and periphery. To gain novel insight into SERT regulation, a comprehensive proteomics screen was conducted to identify components of SERT-associated protein complexes in the brain by employing three independent approaches. In vivo SERT complexes were purified from rat brain using an immobilized high-affinity SERT ligand, amino-methyl citalopram. This approach was combined with GST pulldown and yeast two-hybrid screens using N- and C-terminal cytoplasmic transporter domains as bait. Potential SERT associated proteins detected by at least two of the interaction methods were subjected to gene ontology analysis resulting in the identification of functional protein clusters that are enriched in SERT complexes. Prominent clusters include synaptic vesicle proteins, as well as proteins involved in energy metabolism and ion homeostasis. Using subcellular fractionation and electron microscopy this study provides further evidence that SERT is indeed associated with synaptic vesicle fractions, and colocalizes with small vesicular structures in axons and axon terminals. SERT was also found in close proximity to mitochondrial membranes in both, hippocampal and neocortical regions. A model of the SERT interactome is proposed, in which SERT is distributed between different subcellular compartments through dynamic interactions with site-specific protein complexes. Finally, the protein interaction data suggest novel hypotheses for the regulation of SERT activity and trafficking, which ultimately impact on serotonergic neurotransmission and serotonin dependent brain functions (Haase, 2017).

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, a semi-automated approach was used that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. Antibodies were used against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. A 5-HTergic axon density map is provided, and the ratio is provided of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway (Belmer, 2017).

Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. This study shows that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3 (see Drosophila Serpent), activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3 expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1. Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. This study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation (Haugas, 2016).

Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum

Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). This study visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. It is concluded that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes (Montgomery, 2014).

Search PubMed for articles about Drosophila SerT

Adkins, E. M., Barker, E. L. and Blakely, R. D. (2001). Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition. Mol Pharmacol 59(3): 514-523. PubMed ID: 11179447

Beckman, M. L. and Quick, M. W. (2001). Substrates and temperature differentiate ion flux from serotonin flux in a serotonin transporter. Neuropharmacology 40(4): 526-535. PubMed ID: 11249962

Belmer, A., Klenowski, P. M., Patkar, O. L. and Bartlett, S. E. (2017). Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain. Brain Struct Funct 222(3): 1297-1314. PubMed ID: 27485750

Coleman, J. A. and Gouaux, E. (2018). Structural basis for recognition of diverse antidepressants by the human serotonin transporter. Nat Struct Mol Biol 25(2): 170-175. PubMed ID: 29379174

Condron, B. G. (1999). Serotonergic neurons transiently require a midline-derived FGF signal. Neuron 24(3): 531-540. PubMed ID: 10595507

Couch, J. A., Chen, J., Rieff, H. I., Uri, E. M. and Condron, B. G. (2004). robo2 and robo3 interact with eagle to regulate serotonergic neuron differentiation. Development 131(5): 997-1006. PubMed ID: 14973268

Daubert, E. A., Heffron, D. S., Mandell, J. W. and Condron, B. G. (2010). Serotonergic dystrophy induced by excess serotonin. Mol Cell Neurosci 44(3): 297-306. PubMed ID: 20394820

Davis, B. A., Nagarajan, A., Forrest, L. R. and Singh, S. K. (2016). Mechanism of paroxetine (Paxil) inhibition of the serotonin transporter. Sci Rep 6: 23789. PubMed ID: 27032980

Freissmuth, M., Stockner, T. and Sucic, S. (2018). SLC6 transporter folding diseases and pharmacochaperoning. Handb Exp Pharmacol 245: 249-270. PubMed ID: 29086036

Galli, A., Petersen, C. I., deBlaquiere, M., Blakely, R. D. and DeFelice, L. J. (1997). Drosophila serotonin transporters have voltage-dependent uptake coupled to a serotonin-gated ion channel. J Neurosci 17(10): 3401-3411. PubMed ID: 9133366

Giang, T., Ritze, Y., Rauchfuss, S., Ogueta, M. and Scholz, H. (2011). The serotonin transporter expression in Drosophila melanogaster. J Neurogenet 25(1-2): 17-26. PubMed ID: 21314480

Gu, H. H., Wu, X. and Han, D. D. (2006). Conserved serine residues in serotonin transporter contribute to high-affinity cocaine binding. Biochem Biophys Res Commun 343(4): 1179-1185. PubMed ID: 16580636

Haase, J., Grudzinska-Goebel, J., Muller, H. K., Munster-Wandowski, A., Chow, E., Wynne, K., Farsi, Z., Zander, J. F. and Ahnert-Hilger, G. (2017). Serotonin transporter associated protein complexes are enriched in synaptic vesicle proteins and proteins involved in energy metabolism and ion homeostasis. ACS Chem Neurosci 8(5): 1101-1116. PubMed ID: 28362488

Haugas, M., Tikker, L., Achim, K., Salminen, M. and Partanen, J. (2016). Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe. Development [Epub ahead of print]. PubMed ID: 27789623

Hidalgo, S., Molina-Mateo, D., Escobedo, P., Zarate, R. V., Fritz, E., Fierro, A., Perez, E. G., Iturriaga-Vasquez, P., Reyes-Parada, M., Varas, R., Fuenzalida-Uribe, N. and Campusano, J. M. (2017). Characterization of a Novel Drosophila SERT Mutant: Insights on the Contribution of the Serotonin Neural System to Behaviors. ACS Chem Neurosci 8(10): 2168-2179. PubMed ID: 28665105

Kasture, A., Stockner, T., Freissmuth, M. and Sucic, S. (2017). An unfolding story: Small molecules remedy misfolded monoamine transporters. Int J Biochem Cell Biol 92: 1-5. PubMed ID: 28890376

Kasture, A. S., Hummel, T., Sucic, S. and Freissmuth, M. (2018). Big lessons from tiny flies: Drosophila melanogaster as a model to explore dysfunction of dopaminergic and serotonergic neurotransmitter systems. Int J Mol Sci 19(6). PubMed ID: 29914172

Kasture, A. S., Bartel, D., Steinkellner, T., Sucic, S., Hummel, T. and Freissmuth, M. (2019). Distinct contribution of axonal and somatodendritic serotonin transporters in Drosophila olfaction. Neuropharmacology. PubMed ID: 30851308

Kaufmann, K. W., Dawson, E. S., Henry, L. K., Field, J. R., Blakely, R. D. and Meiler, J. (2009). Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies. Proteins 74(3): 630-642. PubMed ID: 18704946

Larsen, M. B., Elfving, B. and Wiborg, O. (2004). The chicken serotonin transporter discriminates between serotonin-selective reuptake inhibitors. A species-scanning mutagenesis study. J Biol Chem 279(40): 42147-42156. PubMed ID: 15271993

Montgomery, T. R., Steinkellner, T., Sucic, S., Koban, F., Schuchner, S., Ogris, E., Sitte, H. H. and Freissmuth, M. (2014). Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum. J Neurosci 34(18): 6344-6351. PubMed ID: 24790205

Park, S. K., George, R., Cai, Y., Chang, H. Y., Krantz, D. E., Friggi-Grelin, F., Birman, S. and Hirsh, J. (2006). Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT. J Neurobiol 66(5): 452-462. PubMed ID: 16470720

Petersen, C. I. and DeFelice, L. J. (1999). Ionic interactions in the Drosophila serotonin transporter identify it as a serotonin channel. Nat Neurosci 2(7): 605-610. PubMed ID: 10404179

Rodriguez, G. J., Roman, D. L., White, K. J., Nichols, D. E. and Barker, E. L. (2003). Distinct recognition of substrates by the human and Drosophila serotonin transporters. J Pharmacol Exp Ther 306(1): 338-346. PubMed ID: 12682215

Roman, D. L., Saldana, S. N., Nichols, D. E., Carroll, F. I. and Barker, E. L. (2004). Distinct molecular recognition of psychostimulants by human and Drosophila serotonin transporters. J Pharmacol Exp Ther 308(2): 679-687. PubMed ID: 14593087

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date revised: 25 October 2023

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