SLC6 Research Articles

Alternating access of a bacterial homolog of neurotransmitter: sodium symporters determined from AlphaFold2 ensembles and DEER spectroscopy

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understand the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a computational/experimental methodology to illuminate the conformational landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movements of multiple structural motifs that underpin MhsT's conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlights the profound effect of the environment on the energetics of conformational changes. Through experimentally derived selection of collective variables, we present a model of ion and substrate-powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT's function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces an approach that can be applied across a diverse spectrum of transporters to describe their conformational landscapes.

Structure of the human dopamine transporter in complex with cocaine.

The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine1. As part of the neurotransmitter sodium symporter family, DAT uses the Na+ gradient across cell membranes to transport dopamine against its chemical gradient2. The transport mechanism involves both intra- and extracellular gates that control substrate access to a central site. However, the molecular intricacies of this process and the inhibitory mechanism of cocaine have remained unclear. Here, we present the molecular structure of human DAT in complex with cocaine at a resolution of 2.66 Å. Our findings reveal that DAT adopts the expected LeuT-fold, posing in an outward-open conformation with cocaine bound at the central (S1) site. Notably, while an Na+ occupies the second Na+ site (Na2), the Na1 site seems to be vacant, with the side chain of Asn82 occupying the presumed Na+ space. This structural insight elucidates the mechanism for the cocaine inhibition of human DAT and deepens our understanding of neurotransmitter transport. By shedding light on the molecular underpinnings of how cocaine acts, our study lays a foundation for the development of targeted medications to combat addiction.

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT.

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

Unique Substrate Recognition and Sodium–Substrate Binding Stoichiometry in a Bacterial Serotonin Transporter, TuriSERT

All resolved high-resolution structures of the transporters in the neurotransmitter sodium symporter (NSS) family revealed that the NSS members share common structural and mechanistic features for substrate and ion binding and transport. However, a recently reported bacterial orthologue of the human serotonin transporter (hSERT), TuriSERT, possesses a structural characteristic specific for amino acid substrate binding but does transport a biogenic amine. The unique structural feature of TuriSERT requires a novel configuration for coordinating its substrate and ions. In the present study, we characterized TuriSERT expressed in Escherichia coli cells with a fluorescent substrate by biochemical, structural, and pharmacological approaches. Substrate transport by TuriSERT requires Na+ but not Cl−. Replacement of Asp262 by asparagine renders TuriSERT Cl−-dependent. Substitutions of the corresponding Na1 residues did not alter Na+ dependence on substrate transport, whereas the mutation of a Na2 site residue led to a loss of transport activity, suggesting that Na+ binds only to the Na2 site in TuriSERT. In addition, substitutions of several residues essential for recognizing 5-hydroxytryptamine (5-HT) in hSERT had little effect on 5-HT displacement potency in transport assay for TuriSERT. In contrast, mutations of the residues that are proposed to coordinate with 5-HT in our docking model dramatically reduced 5-HT displacement. Furthermore, our results indicated that all tested antidepressants showed a weak inhibitory effect on TuriSERT. The present study demonstrated the existence of a unique substrate binding site and 1:1 stoichiometry of sodium–substrate binding in TuriSERT, a novel structural finding for the NSS transporters.

Carbazole derivatives as promising competitive and allosteric inhibitors of human serotonin transporter: computational pharmacology

The human serotonin transporters (hSERTs) are neurotransmitter sodium symporters of the aminergic G protein-coupled receptors, regulating the synaptic serotonin and neuropharmacological processes related to neuropsychiatric disorders, notably, depression. Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and (S)-citalopram are competitive inhibitors of hSERTs and are commonly the first-line medications for major depressive disorder (MDD). However, treatment-resistance and unpleasant aftereffects constitute their clinical drawbacks. Interestingly, vilazodone emerged with polypharmacological (competitive and allosteric) inhibitions on hSERTs, amenable to improved efficacy. However, its application usually warrants adjuvant/combination therapy, another subject of critical adverse events. Thus, the discovery of alternatives with polypharmacological potentials (one-drug-multiple-target) and improved safety remains essential. In this study, carbazole analogues from chemical libraries were explored using docking and molecular dynamics (MD) simulation. Selectively, two IBScreen ligands, STOCK3S-30866 and STOCK1N-37454 predictively bound to the active pockets and expanded boundaries (extracellular vestibules) of the hSERTs more potently than vilazodone and (S)-citalopram. For instance, the two ligands showed docking scores of −9.52 and −9.59 kcal/mol and MM-GBSA scores of −92.96 and −65.66 kcal/mol respectively compared to vilazodone’s respective scores of −7.828 and −59.27 against the central active site of the hSERT (PDB 7LWD). Similarly, the two ligands also docked to the allosteric pocket (PDB 5I73) with scores of −8.15 and −8.40 kcal/mol and MM-GBSA of −96.14 and −68.46 kcal/mol whereas (S)-citalopram has −6.90 and −69.39 kcal/mol respectively. The ligands also conferred conformational stability on the receptors during 100 ns MD simulations and displayed interesting ADMET profiles, representing promising hSERT modulators for MDD upon experimental validation. Communicated by Ramaswamy H. Sarma

Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1), a member of the neurotransmitter sodium symporter (NSS) family, regulates neuronal excitation by clearing the synaptic cleft of the main inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating GABA levels in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we present the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor, the anticonvulsant tiagabine. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine. Tiagabine's pharmacodynamic profile, confirmed by our experimental data, suggests initial binding to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step induced-fit mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators.

Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [3H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (Kd) for [3H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

Visualizing the binding mode of the antidepressant vilazodone on the serotonin transporter by cryo-EM

The serotonin transporter (SERT) belongs to the family of neurotransmitter sodium symporters (NSS) and is responsible for serotonin recycling and signal termination in the synaptic cleft of neurons. SERT is a therapeutical target for psychiatric disorders such as major depression. The standard medical treatment involves administration of selective serotonin reuptake inhibitors (SSRIs). All investigated SSRIs function as competitive inhibitors to serotonin. However, SERT also possesses an allosteric binding site and allosteric inhibitors may produce a novel therapeutic profile.

Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure–function relationships of NSS family proteins. With hydrogen–deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 – 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.

Glycine Transporter 2: Mechanism and Allosteric Modulation.

Neurotransmitter sodium symporters (NSS) are a subfamily of SLC6 transporters responsible for regulating neurotransmitter signalling. They are a major target for psychoactive substances including antidepressants and drugs of abuse, prompting substantial research into their modulation and structure-function dynamics. Recently, a series of allosteric transport inhibitors have been identified, which may reduce side effect profiles, compared to orthosteric inhibitors. Allosteric inhibitors are also likely to provide different clearance kinetics compared to competitive inhibitors and potentially better clinical outcomes. Crystal structures and homology models have identified several allosteric modulatory sites on NSS including the vestibule allosteric site (VAS), lipid allosteric site (LAS) and cholesterol binding site (CHOL1). Whilst the architecture of eukaryotic NSS is generally well conserved there are differences in regions that form the VAS, LAS, and CHOL1. Here, we describe ligand-protein interactions that stabilize binding in each allosteric site and explore how differences between transporters could be exploited to generate NSS specific compounds with an emphasis on GlyT2 modulation.

Forty Four Years WithBaruch Kanner andThe Chloride Ion.

Baruch Kanner and this author have had parallel careers investigating neurotransmitter transporters. At multiple times during their careers, they have found themselves collaborating or competing, but always learning from each other. This commentary elaborates on the interactions between the Kanner and Rudnick laboratories, with a focus on transporters in the Neurotransmitter: Sodium Symporter (NSS) family of amino acid and amine transporters. A key focus of these interactions is the mechanism by which chloride ions activate and drive transport.

Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer

Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.

Crystal structures of LeuT reveal conformational dynamics in the outward-facing states

The neurotransmitter:sodium symporter (NSS) homolog LeuT from Aquifex aeolicus has proven to be a valuable model for studying the transport mechanism of the NSS family. Crystal structures have captured LeuT in key conformations visited during the transport cycle, allowing for the construction of a nearly complete model of transport, with much of the conformational dynamics studied by computational simulations. Here, we report crystal structures of LeuT representing new intermediate conformations between the outward-facing open and occluded states. These structures, combined with binding and accessibility studies, reveal details of conformational dynamics that can follow substrate binding at the central substrate binding site (S1) of LeuT in outward-facing states, suggesting a potential competition for direction between the outward-open and outward-occluded states at this stage during substrate transport. Our structures further support an intimate interplay between the protonation state of Glu290 and binding of Na1 that may ultimately regulate the outward-open-to-occluded transition.

Allosterically Linked Binding Sites in Serotonin Transporter Revealed by Single Molecule Force Spectroscopy.

Crystal structures and experiments relying on the tools of molecular pharmacology reported conflicting results on ligand binding sites in neurotransmitter/sodium symporters (NSS). We explored the number and functionality of ligand binding sites of NSS in a physiological setting by designing novel tools for atomic force microscopy (AFM). These allow for directly measuring the interaction forces between the serotonin transporter (SERT) and the antidepressant S-citalopram (S-CIT) on the single molecule level: the AFM cantilever tips were functionalized with S-CIT via a flexible polyethylene glycol (PEG) linker. The tip chemistry was validated by specific force measurements and recognition imaging on CHO cells. Two distinct populations of characteristic binding strengths of S-CIT binding to SERT were revealed in Na+-containing buffer. In contrast, in Li+-containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT. The dissociation rate constant of both binding sites was extracted by varying the dynamics of the force-probing experiments. Competition experiments revealed that the two sites are allosterically coupled and exert reciprocal modulation.

Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS

Neurotransmitter:sodium symporters (NSS) are integral membrane proteins (IMP), responsible for reuptake of neurotransmitters from the synaptic cleft. Due to challenges in production of mammalian NSS in their active form, the prokaryotic hydrophobic amino acid transporter, LeuT, served here as a steadfast model for elucidation of structure-function relationship. As NSS proteins reside within phospholipid bilayer, they require stabilization by artificial membrane systems upon their extraction. Right choice of artificial membrane system is crucial as suboptimal detergent and/or lipids can lead to destabilization or non-native stabilization. Here we study the effect of related detergents, dodecyl maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG), on the conformational dynamics of LeuT by global HDX-MS, in the presence of functionally relevant ligands. We observed that LeuT is more dynamic when solubilized in DDM compared to LMNG. Moreover, LeuT exhibited increased HDX in the presence of K+ compared to Na+, indicating a more dynamic conformation in the presence of K+. Upon addition of leucine, LeuT underwent additional stabilization relative to the Na+-bound state. Finally, peak broadening was observed, suggesting that LeuT undergoes slow unfolding/refolding dynamics in detergent solution. These slow dynamics were verified by local HDX, also proving that detergents modulate the rate of these dynamics. SignificanceOverall, we show the efficacy of global HDX-MS to evaluate the effect of artificial membrane systems on integral membrane proteins and the importance of carefully selecting compatible detergent (and/or lipid) for the solubilization of this class of proteins.

X-ray structure of LeuT in an inward-facing occluded conformation reveals mechanism of substrate release

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins.

A Kinetic Description of Cytosolic K+ Binding to the Human Sertotonin Transporter Under Turnover Conditions

The human serotonin transporter (hSERT) belongs to the neurotransmitter:sodium symporter (NSS) family, which regulate neurotransmission by re-uptaking the released neurotransmitters into the presynaptic neuron. During the transport cycle, the transporter alternates between the outward-facing (OF) and inward-facing (IF) states to translocate the substrate across the membrane. Central to the cycle in hSERT is the OF-to-IF state transition, during which 5-HT (serotonin) is inwardly symported in a Na+- and Cl--dependent manner, followed by cytosolic-K+-binding-triggered returning to the OF state. K+ binding might act as a “kinetic decision point” by frustrating the outward transport of 5-HT, namely, the reverse of the physiological direction. However, the mechanism of such K+ regulation remains elusive. Moreover, the conventional transport stoichiometry (15-HTin:1Na+in:1Cl−in:1K+out) has been challenged by recent studies showing that Cl- might remain bound to hSERT during the entire cycle. To explore the role of cytosolic K+ binding to IF hSERT, we have constructed a Markov State Model from an extensive set of molecular dynamics simulations. Starting with the IF state, the system is modeled under turnover conditions, i.e., post-5-HT-release and exposed to cytosolic K+ concentration, resulting in 50 independent, 200-ns replicas. More than half of the trajectories capture spontaneous K+ binding to the Na2 site, a conserved Na+ binding site within NSSs deemed to control the transporter conformational changes and substrate release. Furthermore, consistent with recent experimental results, Cl- was found to remain bound in the majority of the ensembles, questioning the contribution of the Cl- gradient to the substrate transport. Together, these results characterize the previously unknown K+ site in hSERT and present a quantitative kinetic description of K+ binding, shedding light on the mechanism of the ion-dependence of the IF-to-OF transition that resets the transport cycle.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

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. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe 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 ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a 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 detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.