SLC6 Research Articles

Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport.

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons1-3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action2,4. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines2,3, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder2,5. Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6-12. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties13,14. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors15,16. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

Substrate-Induced Conformational Dynamics of the Dopamine Transporter

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. The coordinated conformational rearrangements related to the transport mechanism have so far been poorly understood. Here we have probed the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect novel ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a novel molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing the first detailed insight into the state-dependent conformational changes associated with the alternating access model of a eukaryotic NSS.

Transporter oligomerisation: roles in structure and function.

Oligomerisation is a key feature of integral membrane transporters with roles in structure, function and stability. In this review, we cover some very recent advances in our understanding of how oligomerisation affects these key transporter features, with emphasis on a few groups of transporters, including the nucleobase ascorbate transporters, neurotransmitter sodium symporters and major facilitator superfamily members.

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT in Escherichia coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

Exploring Substrate Binding in the Extracellular Vestibule of MhsT by Atomistic Simulations and Markov Models.

Neurotransmitter:sodium symporters (NSS) terminate neurotransmission through Na+-driven reuptake of cognate neurotransmitters. Crystallographically, whereas both substrates and inhibitors have been found to bind in the central binding (S1) site of NSS, inhibitors were found to bind to a second binding (S2) site in the extracellular vestibule (EV) of transporters for leucine (LeuT) and serotonin. On the basis of computational and experimental studies, we proposed that substrates bind to the S2 site of LeuT as well and that substrate binding to the S2 site is essential for Na+-coupled symport. Recent binding experiments show that substrate (l-Trp) binding in the S2 site of MhsT, another bacterial NSS, is also central to the allosteric transport mechanism. Here, we used extensive molecular dynamics simulations combined with Markov state model analysis to investigate the interaction of l-Trp with the EV of MhsT and identified potential binding poses of l-Trp as well as induced conformational changes in the EV. Our computational findings were validated by experimental mutagenesis studies and shed light on the ligand binding characteristics of the EV of NSS, which may facilitate development of allosteric ligands targeting NSS.

Elucidation of self‐mediated enhancement of dopamine transport by the dopamine transporter which can be modulated by extracellular gate and N‐terminal residues

The dopamine (DA) and serotonin (5‐HT) transporters (DAT and SERT respectively) are members of the SLC6 family of neurotransmitter:sodium symporters (NSSs). DAT and SERT clear substrate from perisynaptic spaces by the generally accepted alternating access mechanism in which the transporters transition between outward‐facing and inward‐facing conformational states. This mechanism involves a sequential closing and opening of extracellular (EC) and intracellular (IC) gates along the permeation pathway. The EC and IC gates are almost exclusively composed of acidic and basic residues capable of interacting through salt bridge formation, closing the primary substrate binding site to the intracellular and extracellular milieus. Previously, we identified that the human SERT acidic/basic residue pair is critical for the recognition of 3,4‐methylenedioxymethamphetamine (MDMA) as a substrate. In contrast, mutations in the EC gate had no effect on 5‐HT recognition. Our current efforts focus on determining if the EC gate of the human DAT has similar mechanistic characteristics for substrate selectivity. While investigating that possibility, we identified a previously unreported phenomenon of self‐mediated enhancement of DA transport in hDAT expressing cells. In homologous competitive uptake experiments utilizing [3H]DA, a 200% to 300% increase in [3H]DA uptake is observed upon addition of micromolar concentrations of unlabeled DA. This enhancement is further modulated by the identity of the acidic EC gate residue. Substituting the Asp with the polar amino acid Asn (D476N) produces a 600% increase in [3H]DA uptake. Experiments utilizing heterologous competition with [3H]DA and the known hDAT substrates 1‐methyl‐4‐phenylpyridinium (MPP+), 4‐(4‐dimethylamino)phenyl‐1‐methylpyridinium(APP+) and tyramine indicate that the enhancement effect is not exclusive to DA nor is it observed with all DAT substrates. Notably, elimination of phosphorylation at N‐terminal residue Thr53 virtually eliminates the DA self‐enhancement phenotype. The apparent ability of DA to enhance its own uptake is consistent with reports proposing a second substrate binding site (S2) that can act as a trigger for release of substrate from the primary DA binding site (S1). However, the presence of this S2 site remains controversial. Continued investigation of this phenomenon may provide insight into molecular determinants on both the substrate and transporter that are important for substrate identification and translocation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thermodynamic Coupling Function Analysis of Allosteric Coupling between Na+ Release and Inward-Opening in the Human Dopamine Transporter

Allostery plays a crucial role in the mechanism of neurotransmitter-sodium symporters, such as the human dopamine transporter (hDAT). To investigate the molecular mechanism that couples transport-associated inward release of Na+ from the Na2 site to conformational changes associated with inward-opening, we applied a novel combination of our recently developed thermodynamic coupling function (TCF) theory of allostery and Markov State Model (MSM) analysis to a 50-microsecond dataset of Molecular Dynamics trajectories of hDAT, in which multiple spontaneous Na+ release events were observed. Our TCF approach reveals a complex landscape of thermodynamic coupling between Na+ release and inward-opening, and identifies diverse, yet well-defined roles for different Na+-coordinating residues. In particular, we identify a prominent role in the allosteric coupling for the Na+-coordinating residue D421, where mutation has previously been associated with neurological disorders. Our results highlight the power of the TCF theory and analysis to elucidate the molecular mechanism of complex allosteric processes in large biomolecular systems.

A partially-open inward-facing intermediate conformation of LeuT is associated with Na+ release and substrate transport

Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.

Two Ligand Binding Sites in Serotonin Transporter Revealed by Nanopharmacological Force Sensing.

The number of ligand binding sites in neurotransmitter-sodium symporters has been determined by crystal structure analysis and molecular pharmacology with controversial results. Here, we designed molecular tools to measure the interaction forces between the serotonin transporter (SERT) and S-citalopram on the single-molecule level by means of atomic force microscopy. Force spectroscopy allows for the extraction of dynamic information under physiological conditions which is inaccessible via X-ray crystallography. Two populations of distinctly different binding strength between S-citalopram and SERT were demonstrated in Na+-containing buffer. In Li+-containing buffer, SERT showed merely low-force interactions, whereas the vestibular mutant SERT-G402H only displayed the high force population. These observations provide physical evidence for the existence of two different binding sites in SERT when tested under near-physiological conditions.

Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter

Allostery plays a crucial role in the mechanism of neurotransmitter-sodium symporters, such as the human dopamine transporter. To investigate the molecular mechanism that couples the transport-associated inward release of the Na+ ion from the Na2 site to intracellular gating, we applied a combination of the thermodynamic coupling function (TCF) formalism and Markov state model analysis to a 50-μs data set of molecular dynamics trajectories of the human dopamine transporter, in which multiple spontaneous Na+ release events were observed. Our TCF approach reveals a complex landscape of thermodynamic coupling between Na+ release and inward-opening, and identifies diverse, yet well-defined roles for different Na+-coordinating residues. In particular, we identify a prominent role in the allosteric coupling for the Na+-coordinating residue D421, where mutation has previously been associated with neurological disorders. Our results highlight the power of the TCF analysis to elucidate the molecular mechanism of complex allosteric processes in large biomolecular systems.

Identification of novel serotonin reuptake inhibitors targeting central and allosteric binding sites: A virtual screening and molecular dynamics simulations study

The serotonin (5-hydroxytryptamine, 5HT) transporter (SERT) is a member of neurotransmitter sodium symporter (NSS) family, which maintains neurotransmitter by reuptaking 5HT into synapses. Decrease in serotonin concentrations in synaptic clefts have been reported to cause psychological and neurological disorders. Therefore, inhibition of SERT is a potent strategy for the treatment of related diseases such as depression. In this study, approximately 260,000 small molecules from an available chemical database have been virtually screened both at central and allosteric binding sites of SERT to identify potent novel candidate SERT inhibitors. A set of docking algorithms were used to predict binding modes and energies of compounds. Screening analyses led three top-ranked hit compounds (160234, Otava ID: 7118020138; 159166, Otava ID: 7117171303; and 69419, Otava ID: 118671819) for central binding site (S1) and one compound (93507, Otava ID: 6248262) for allosteric binding site (S2). These promising compounds are then subjected to long multiple molecular dynamics (MD) simulations to elucidate their structural and dynamical profiles at the binding cavities of SERT. Higher predicted binding affinities of identified compounds were also confirmed with binding free energy calculations (MM/GBSA) in comparison with the reference central and allosteric binding site inhibitors, paroxetine (8PR) and escitalopram (68P), respectively. To the best of our knowledge, the present work is the first structure-based high throughput virtual screening study reported using recently revealed crystal structure of SERT for screening inhibitors from chemical databases on S1 and S2 binding sites. Small molecule library screening study yielded candidate compounds both at central and allosteric binding site of SERT, and further experimentation may pave the way for developing novel strong inhibitors.

The role of transmembrane segment 5 (TM5) in Na2 release and the conformational transition of neurotransmitter:sodium symporters toward the inward-open state

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by the reuptake of released neurotransmitters. This active accumulation of substrate against its concentration gradient is driven by the transmembrane Na+ gradient and requires that the transporter traverses several conformational states. LeuT, a prokaryotic NSS homolog, has been crystallized in outward-open, outward-occluded, and inward-open states. Two crystal structures of another prokaryotic NSS homolog, the multihydrophobic amino acid transporter (MhsT) from Bacillus halodurans, have been resolved in novel inward-occluded states, with the extracellular vestibule closed and the intracellular portion of transmembrane segment 5 (TM5i) in either an unwound or a helical conformation. We have investigated the potential involvement of TM5i in binding and unbinding of Na2, i.e. the Na+ bound in the Na2 site, by carrying out comparative molecular dynamics simulations of the models derived from the two MhsT structures. We find that the helical TM5i conformation is associated with a higher propensity for Na2 release, which leads to the repositioning of the N terminus and transition to an inward-open state. By using comparative interaction network analysis, we also identify allosteric pathways connecting TM5i and the Na2 binding site to the extracellular and intracellular regions. Based on our combined computational and mutagenesis studies of MhsT and LeuT, we propose that TM5i plays a key role in Na2 binding and release associated with the conformational transition toward the inward-open state, a role that is likely to be shared across the NSS family.

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.

Effect of Dimerization on the Dynamics of Neurotransmitter:Sodium Symporters.

Dimerization is a common feature among the members of the neurotransmitter:sodium symporter (NSS) family of membrane proteins. Yet, the effect of dimerization on the mechanism of action of NSS members is not fully understood. In this study, we examined the collective dynamics of two members of the family, leucine transporter (LeuT) and dopamine transporter (DAT), to assess the significance of dimerization in modulating the functional motions of the monomers. We used to this aim the anisotropic network model (ANM), an efficient and robust method for modeling the intrinsic motions of proteins and their complexes. Transporters belonging to the NSS family are known to alternate between outward-facing (OF) and inward-facing (IF) states, which enables the uptake and release of their substrate (neurotransmitter) respectively, as the substrate is transported from the exterior to the interior of the cell. In both LeuT and DAT, dimerization is found to alter the collective motions intrinsically accessible to the individual monomers in favor of the functional transitions (OF ↔ IF), suggesting that dimerization may play a role in facilitating transport.

Exploring Ligand-Binding Kinetics in the S2 Site of MhsT by Atomistic Simulations and Markov Models

Neurotransmitter:Sodium Symporters (NSS) terminate neurotransmission through sodium-driven reuptake of cognate neurotransmitters, and traverse between outward-open and inward-open states. NSS include serotonin and dopamine transporters, which are targets for antidepressants and abused psychostimulants. Crystallographically, whereas both substrates and inhibitors have been found to bind the central binding site (denoted S1) of the NSS proteins, it has been shown that inhibitors for LeuT and SERT can also bind to a binding cavity in the extracellular vestibule (denoted S2). Based on computational and experimental studies in LeuT, it has been found that substrates can bind to S2 as well, and such binding triggers the intracellular release the substrate and Na+ from S1. However, whether such a role of S2 substrate is common for other NSS remains unclear.The newly available crystal structure of MhsT, a bacterial NSS homolog, which was solved in an inward-occluded state, provides a more suitable starting point to study the substrate induced conformational changes, including the S2 substrate induced transition from inward-occluded to inward-open state transition. Here, we use extensive molecular dynamics simulations combined with hidden Markov model (HMM) analysis to investigate the feasibility and kinetics of the substrate L-tryptophan binding to S2 of MhsT. Based on HMM analysis, we identified transition binding positions in the extracellular vestibule that form potential substrate binding pathways, and the conformational changes near the S2 site associated with the two most populated bound states. Our findings shed light on the S2 substrate binding kinetics and the induced conformational changes, which may be critical in triggering the subsequent events in the transport mechanism.

Characterizing Outward- to Inward-Facing Transition Pathway of Dopamine Transporter

The dopamine transporter (DAT) belongs to the family of neurotransmitter sodium symporters (NSSs), which harness transmembrane electrochemical ionic gradients to transport neurotransmitters against their chemical gradients. Dysregulation of DAT is associated with serious neurological disorders, such as Parkinson's disease, depression, anxiety and epilepsy. As with all transporters, substrate translocation through DAT follows the alternating-access mechanism in which the protein swiches between outward-facing (OF) and inward-facing (IF) states. The details of these structural changes and their coupling to chemical events such as substrate and ion binding events remain elusive. In the present study, we charactrized large-scale transition from the OF state to IF state using all-atom molecular dynamics simulations of membrane-bound models of DAT. As all crystal structures of DAT are in the OF state, the initial phase of the study included modeling of a stable IF structure of DAT in the context of a membrane using the bacterial sodium-coupled leucine symporter (LeuT) as a template. Furthermore, equilibrium simulations performed in this phase revealed a novel sodium binding site located between TM3 and TM8 helices, which are elements involved in coupling of protein structural changes to substrate binding and translocation. Using the orientations of helics TM1e/TM8e and TM1i/TM8i as collective variables, and employing two dimensional bias-exchange umbrella sampling and string method with swarms of trajectories, we characerize a structural transition pathway between the OF and IF states of DAT. The results of this study provide a deeper understanding of the functional mechanism of DAT, wiht implications to all members of the NSS family.

Markov State-Based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

The dopamine transporter (DAT) belongs to the neurotransmitter/sodium symporter (NSS) family of membrane proteins that are responsible for reuptake of neurotransmitters from the synaptic cleft to terminate a neuronal signal and enable subsequent neurotransmitter release from the presynaptic neuron. The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identified as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process are computed from the MSM, and transition path theory is used to identify the most probable sodium release pathways. An intermediate state is discovered on the sodium release pathway, and the results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.

Identification of Fluoxetine-Serotonin Transporter Interactions using Crosslinking-Mass Spectrometry (CX-MS)

The serotonin transporter (SERT) is a member of the neurotransmitter sodium symporter family of transporters (NSSs). SERT controls the magnitude and duration of serotonergic neurotransmission by facilitating the reuptake of serotonin back into the pre-synaptic neuron and is thus a target for antidepressants and selective serotonin re-uptake inhibitors (SSRIs) commonly prescribed to treat depression. To understand drug binding to SERT, a photo-activatable fluoxetine analog was synthesized and crosslinked to human SERT expressed in tetracycline-inducible HEK293 cells. After purification, trypsinized peptides were analyzed by ESI-Q-TOF and MS/MS studies identified sites of covalent attachment. Given that fluoxetine partitions in bilayers and might interact non-specifically with membrane proteins, control studies with photo-activatable lipids were also conducted to map lipid accessible sites. The hSERT expression system contains N-terminal tags for purification, so additional CX-MS studies were conducted using baculovirus overexpression of rat SERT containing analogous tags, but at its C-terminus to control for artefacts attributable to the sites of the chimeric tags. Altogether, these studies identify non-specific and specific sites of interactions between fluoxetine and SERT.

Direct assessment of substrate binding to the Neurotransmitter:Sodium Symporter LeuT by solid state NMR.

The Neurotransmitter:Sodium Symporters (NSSs) represent an important class of proteins mediating sodium-dependent uptake of neurotransmitters from the extracellular space. The substrate binding stoichiometry of the bacterial NSS protein, LeuT, and thus the principal transport mechanism, has been heavily debated. Here we used solid state NMR to specifically characterize the bound leucine ligand and probe the number of binding sites in LeuT. We were able to produce high-quality NMR spectra of substrate bound to microcrystalline LeuT samples and identify one set of sodium-dependent substrate-specific chemical shifts. Furthermore, our data show that the binding site mutants F253A and L400S, which probe the major S1 binding site and the proposed S2 binding site, respectively, retain sodium-dependent substrate binding in the S1 site similar to the wild-type protein. We conclude that under our experimental conditions there is only one detectable leucine molecule bound to LeuT.

A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

The dopamine transporter (DAT) belongs to the neurotransmitter:sodium symporter (NSS) family of membrane proteins that are responsible for reuptake of neurotransmitters from the synaptic cleft to terminate a neuronal signal and enable subsequent neurotransmitter release from the presynaptic neuron. The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identified as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process are computed from the MSM, and transition path theory is used to identify the most probable sodium release pathways. An intermediate state is discovered on the sodium release pathway, and the results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.