The serotonin transporter (SERT) belongs to the family of neurotransmitter sodium symporters (NSS), together with other neurotransmitter transporters for norepinephrine, dopamine, glycine, and GABA. The main physiological role of SERT is the retrieval of previously released serotonin from the synaptic cleft. Thereby, SERT plays an important role in regulating the extracellular serotonin concentration and maintaining serotonergic neurotransmission. This process can be influenced by molecules acting as serotonin uptake inhibitors, like paroxetine. Here, we report the development of a novel photoswitchable paroxetine derivative and its pharmacological interaction profile with SERT as a tool compound for the light-induced control of SERT. Based on the azo-extension strategy, the photoswitchable moiety was formed at the former position of the fluoro substituent in paroxetine. The resulting azo-paroxetine (9) was easily and reversibly switched between active (Z) and inactive (E) configurations and remained stable between these configurations: serotonin uptake was inhibited more than 12 times more potently by the active (Z)-configuration having a sub μM IC50 value. This was supported by electrophysiological patch-clamp recordings in the whole-cell configuration and docking studies. No significant toxic impact of azo-paroxetine (9) and no off-target activity at the norepinephrine transporter (NET), human GABA transporter subtypes 1 and 3, and rat GAT1 were observed. Our results demonstrate that the activity of SERT can be reversibly manipulated by the optopharmacological agent azo-paroxetine (9). This compound can thus be applied as a tool for the selective manipulation of SERT in central or peripheral investigations, further benefiting from its low probability for compound-related off-target effects.

The human serotonin transporter SERT facilitates serotonin (5-HT+) transport into cells by coupling to Na+ co-transport and K+ exchange. Although extracellular Cl- is also essential for transport, whether Cl- ions are transported has been disputed, raising the question why Cl- ions are required? Here, we examine the role of Cl- using transport measurements, conformational assays, and molecular simulations. We show that Cl- is not transported and does not affect Na+-mediated cytoplasmic pathway closure but does reduce the accessibility of residues in the extracellular pathway, mimicking transport-related occlusion. Simulations indicate that Cl- ion binding constrains the helices in the so-called bundle, but not interactions spanning the extracellular pathway thought to act as a molecular gate. We surmise that Cl- (i) increases the stability of surrounding helices, (ii) enhances Na+ binding affinity, and (iii) decreases extracellular pathway accessibility, thereby facilitating transport-related conformational changes. These findings explain SERT's requirement for chloride and highlight distinct features of proteins in the same neurotransmitter transporter family.

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter SERT, which couples this process to the movement of a Na+ion down its chemical gradient. After release of 5-HT and Na+into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+binding. Early studies of vesicles containing native SERT revealed that K+gradients can provide an additional driving force, via K+antiport. Moreover, under appropriate conditions, a H+ion can replace K+. Intracellular K+accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ions, but the K+site remains enigmatic. Here, we show that K+antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+binds to the so-called Na2+site. Site directed mutagenesis of residues in this site can eliminate the ability of both K+and H+to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+or H+. In conclusion, the Na2+site plays a pivotal role in orchestrating the sequential binding of Na+and then K+(or H+) ions to facilitate 5-HT uptake in SERT.

The human serotonin transporter hSERT, or SLC6A4, from the solute carrier 6 (SLC6) protein family, plays an important role in the central and peripheral nervous systems. Its major neurobiological task is the termination of serotonergic neurotransmission by reuptake of its endogenous substrate, serotonin, into presynaptic boutons after signaling. The transporter undergoes several conformational changes in each physiological substrate uptake cycle and interacts with protons, sodium, chloride, and potassium ions during these transitions. Over the years, a substantial body of knowledge about hSERT function and mechanism has been accumulated, significantly improving pharmacological treatment options especially for major depression by covering several chemical compound classes. Nevertheless, effective symptom amelioration, side effect profiles, and patient compliance are still far from ideal and require urgent action. For this reason, a fundamental approach is to tackle key unanswered questions regarding the transporter's function, including the mechanism of potassium and proton binding, whose roles are both interconnected and poorly understood. To this end, we have applied molecular simulation approaches at the coarse-grained and fully-atomistic levels, including free energy calculations. The simulations predict the locations of the potassium and proton binding sites and suggest the molecular origins of selectivity at each site. In addition to the gains in basic scientific understanding, the knowledge obtained paves the way for designing mechanism-based transporter ligands that are useful as highly specialized therapeutic agents.

The transporters for dopamine (DAT) and serotonin (SERT) are important targets in the treatment of psychiatric disorders including major depression, anxiety and attention-deficit hyperactivity disorder. Drugs acting at these transporters can act as inhibitors or as releasers. In addition, it has been recently appreciated that some compounds are less efficacious releasers than amphetamine. Thus, they are classified as partial releasers. Compounds can act on both SERT and DAT or display exquisite selectivity for either SERT or DAT, but the structural basis for selectivity is poorly understood. The trifluoromethyl-substitution of methcathinone in the para-position has been shown to dramatically shift the selectivity of methcathinone (MCAT) towards SERT. Here, we examined MCAT, para-trifluoromethyl-methcathinone (pCF3MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF3-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the pCF3group, but not of other substituents with larger van der Waal's volume, lipophilicity or polarity, converted the DAT-selective MCAT into a SERT-selective partial releaser. Electrophysiological and superfusion experiments, together with kinetic modelling, showed that pCF3MCAT, but not MCAT, trapped a fraction of SERTs in an inactive state by occupying the S2-site. These findings define a new mechanism of action for partial releasers, which is distinct from the other two known binding modes underlying partial release. Our observations highlight the fact that the substrate permeation pathway of monoamine transporters supports multiple binding modes, which can be exploited for drug design. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Methcathinone (MCAT) is a compound belonging to the class of cathinones which targets monoamine transporters including DAT and SERT. Despite the importance of DAT and SERT as drug targets in several neurological disorders, the key factors underlying the selectivity profiles of their inhibitors are still poorly understood. Recent findings from rat synaptosomes suggested that increasing the volume of the para‐substituent of MCAT results in a swap of the selectivity between human DAT and SERT (1). Docking studies hint towards Ser149 in DAT and Ala169 in SERT as key residues involved in the difference of activity between DAT and SERT (2). The aims of the present biochemical and pharmacological study are to understand (i) which chemical properties (e.g. volume, polarity, or lipophilicity) of the para‐substituent influence the selectivity profile of MCAT between DAT and SERT, and (ii) whether Ser149 in DAT and Ala169 in SERT can be experimentally verified as key residues. Hence, we combined in silico‐driven synthesis, mutagenesis, and radio‐tracer flux assays in HEK293 cells expressing the human DAT and SERT wild‐type and respective mutants. We found that only MCAT and the para‐CF3‐MCAT showed high selectivity, 200‐fold for DAT/SERT ‐ and 25‐fold for SERT/DAT, respectively. This suggests that the high selectivity achieved is determined rather by specific features of these compounds than by the volume of the para‐substituent. Accordingly, we were not able to find any correlation between the selectivity profile of the tested 4MCATs with either volume, polarity, or lipophilicity parameters. Next, based on our results, we tested the hypothesis that the two amino acids in SERT and DAT do not suffice to explain the selectivity between SERT and DAT. We have tested the para‐substituted methcathinones in the swapping mutations DAT Ser149Ala and SERT Ala169Ser and in line with our hypothesis, these mutations did not revert the selectivity profile found in the wild‐type transporters. Our findings rather suggest that the SERT selectivity achieved by the introduction of the CF3‐group in the para‐position of MCAT is not dependent on the volume of the para‐substituent but on specific chemical features of the fluorine atoms which may influence the on and off rate of the MCAT molecule on DAT and SERT.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

ɣ-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

The human monoamine transporters play an important role in the central and peripheral nervous system by regulating signals among neurons. Numerous compound classes have been identified to interact with these proteins, either used in a therapeutic setting or abused as illicit drugs. However, based on the physicochemical properties of these ligands alone, the mechanistic distinction between transporter substrates and inhibitors is not always clear. For example, it remains to be established whether the closure of the extracellular pathway facilitated by substrate can also occur when such inhibitors are bound. It therefore seems necessary to pinpoint the molecular features of the substrate required for the transport-related conformational change, and in particular, the outward-open to outward-occluded transition. A recently-reported structure of the outward-open state of human serotonin transporter (hSERT), provides a starting point for molecular dynamics (MD) simulations of this transition. Initial MD simulations revealed stability and convergence of this conformation over time. However, the endpoint of this pathway closure event is not yet known. Therefore, we present a carefully-refined homology model of hSERT based on a combination of two templates (hSERT, outward-open and the bacterial leucine transporter LeuT, outward-occluded) in an outward-occluded state. This homology model will be used as the endpoint of enhanced sampling simulations of the transition between the outward-open and the outward-occluded state of hSERT. Combined with knowledge about substrates and inhibitors sharing the same scaffold interacting with this transporter, our approaches will lead to a better understanding of the transporter mechanism.

Correction for 'From linked open data to molecular interaction: studying selectivity trends for ligands of the human serotonin and dopamine transporter’ by Barbara Zdrazil et al., Med. Chem. Commun., 2016, 7, 1819–1831.

ATP-driven transport across biological membranes is a key process to translocate solutes from the interior of the cell to the extracellular environment. In humans, ATP-binding cassette transporters are involved in absorption, distribution, metabolism, excretion, and toxicity, and also play a major role in anticancer drug resistance. Analogous transporters are also known to be involved in phytohormone translocation. These include, e.g., the transport of auxin by ABCB1/19 in Arabidopsis thaliana, the transport of abscisic acid by AtABCG25, and the transport of strigolactone by the Petunia hybrida ABC transporter PDR1. Within this article, we outline the current knowledge about plant ABC transporters with respect to their structure and function, and provide, for the first time, a protein homology model of the strigolactone transporter PDR1 from P. hybrida.