Identification of the potassium-binding site in serotonin transporter.

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.

A structural model of the human serotonin transporter in an outward-occluded state.

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.

Structural and molecular aspects of betaine-GABA transporter 1 (BGT1) and its relation to brain function.

É£-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'.

para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter.

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'.

From linked open data to molecular interaction: studying selectivity trends for ligands of the human serotonin and dopamine transporter.

Retrieval of congeneric and consistent SAR data sets for protein targets of interest is still a laborious task to do if no appropriate in-house data set is available. However, combining integrated open data sources (such as the Open PHACTS Discovery Platform) with workflow tools now offers the possibility of querying across multiple domains and tailoring the search to the given research question. Starting from two phylogenetically related protein targets of interest (the human serotonin and dopamine transporters), the whole chemical compound space was explored by implementing a scaffold-based clustering of compounds possessing biological measurements for both targets. In addition, potential hERG blocking liabilities were included. The workflow allowed studying the selectivity trends of scaffold series, identifying potentially harmful compound series, and performing SAR, docking studies and molecular dynamics (MD) simulations for a consistent data set of 56 cathinones. This delivered useful insights into driving determinants for hDAT selectivity over hSERT. With respect to the scaffold-based analyses it should be noted that the cathinone data set could be retrieved only when Murcko scaffold analyses were combined with similarity searches such as a common substructure search.

The ABC of Phytohormone Translocation.

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.