Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na⁺/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport.     

Properties of an Inward-Facing State of LeuT: Conformational Stability and?Substrate Release

The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na⁺ ions and substrate bound suggest that one of the Na⁺ ion binding sites is fully disrupted. Release of alanine and the second Na⁺ ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.     

Substrate binding and formation of an occluded state in the leucine transporter

Translocation through the extracellular vestibule and binding of leucine in the leucine transporter (LeuT) have been studied with molecular dynamics simulations. More than 0.1 μs of all-atom molecular dynamics simulations have been performed on different combinations of LeuT, bound substrate, and bound structural Na⁺ ions to describe molecular events involved in substrate binding and in the formation of the occluded state and to investigate the dynamics of this state. Three structural features are found to be directly involved in the initial steps of leucine transport: a Na⁺ ion directly coordinated to leucine (Na-1), two aromatic residues closing the binding site toward the extracellular vestibule (Tyr-108 and Phe-253), and a salt bridge in the extracellular vestibule (Arg-30 and Asp-404). These features account for observed differences between simulations of LeuT with and without bound substrate and for a possible pathway for leucine binding and thereby formation of the occluded LeuT binding site.     

Ion-Controlled Conformational Dynamics in the Outward-Open Transition from an Occluded State of LeuT

Neurotransmitter:sodium symporter (NSS) proteins are secondary Na⁺-driven active transporters that terminate neurotransmission by substrate uptake. Despite the availability of high-resolution crystal structures of a bacterial homolog of NSSs—Leucine Transporter (LeuT)—and extensive computational and experimental structure-function studies, unanswered questions remain regarding the transport mechanisms. We used microsecond atomistic molecular-dynamics (MD) simulations and free-energy computations to reveal ion-controlled conformational dynamics of LeuT in relation to binding affinity and selectivity of the more extracellularly positioned Na⁺ binding site (Na1 site). In the course of MD simulations starting from the occluded state with bound Na⁺, but in the absence of substrate, we find a spontaneous transition of the extracellular vestibule of LeuT into an outward-open conformation. The outward opening is enhanced by the absence of Na1 and modulated by the protonation state of the Na1-associated Glu-290. Consistently, the Na⁺ affinity for the Na1 site is inversely correlated with the extent of outward-open character and is lower than in the occluded state with bound substrate; however, the Na1 site retains its selectivity for Na⁺ over K⁺ in such conformational transitions. To the best of our knowledge, our findings shed new light on the Na⁺-driven transport cycle and on the symmetry in structural rearrangements for outward- and inward-open transitions.     

Insights on Na+ binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+‐substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild‐type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na⁺ ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di‐Na⁺ binding state is likely more efficient for conformational changes of NorM_VC toward the inward‐facing conformation than single‐Na⁺ binding state. The observation of two Na⁺ binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na⁺ binding and enrich our knowledge of the transport mechanism of secondary transporters. Proteins 2014; 82:240–249. © 2013 Wiley Periodicals, Inc.     

Molecular mechanism of ion-ion and ion-substrate coupling in the Na+-dependent leucine transporter LeuT

Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high-resolution structure for a bacterial homolog of Na⁺/Cl⁻-dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only ∼7 Å away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na⁺ and not to Li⁺ or K⁺ cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na⁺ in the case of double ion occupancy.     

Molecular dynamics of leucine and dopamine transporter proteins in a model cell membrane lipid bilayer

The dopamine transporter (DAT) operates via facilitated diffusion, harnessing an inward Na⁺ gradient to drive dopamine from the extracellular synaptic cleft to the neuron interior. The DAT is relevant to central nervous system disorders such as Parkinson disease and attention‐deficit hyperactivity disorder and is the primary site of action for the abused psychostimulants cocaine and amphetamines. Crystallization of a DAT homolog, the bacterial leucine transporter LeuT, provided the first reliable 3‐D DAT template. Here, the LeuT crystal structure and the DAT molecular model have been combined with their respective substrates, leucine and dopamine, in lipid bilayer molecular dynamics simulations toward tracking substrate movement along the protein's substrate/ion permeation pathway. Specifically, movement of residue pairs that comprise the “external gate” was followed as a function of substrate presence. The transmembrane (TM) 1 arginine‐TM 10 aspartate strut formed less readily in DAT compared with LeuT, with or without substrate present. For LeuT but not DAT, the addition of substrate enhanced the chances of forming the TM 1‐10 bridge. Also, movement of the fourth extracellular loop EL‐4 in the presence of substrate was more pronounced for DAT, the EL‐4 unwinding to a degree. The overall similarity between the LeuT and DAT molecular dynamics simulations indicated that LeuT was a legitimate model to guide DAT structure‐function predictions. There were, nevertheless, differences significant enough to allow for DAT‐unique insights, which may include how cocaine, methylphenidate (Ritalin, NIDA Drug Supply, Rockville, MD), and other DAT blockers are not recognized as substrates even though they can access the primary substrate binding pocket. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms

The x-ray structure of LeuT, a bacterial homologue of Na⁺/Cl⁻-dependent neurotransmitter transporters, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion binding sites, NA1 and NA2, which are highly selective for Na⁺. Extensive all-atom free-energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na⁺ over K⁺ or Li⁺, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In NA1, selectivity for Na⁺ over K⁺ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In NA2, which comprises only neutral ligands, selectivity for Na⁺ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the polypeptide chain surrounding the ion according to a “snug-fit” mechanism.     

Simulated annealing reveals the kinetic activity of SGLT1, a member of the LeuT structural family

The Na⁺/glucose cotransporter (SGLT1) is the archetype of membrane proteins that use the electrochemical Na⁺ gradient to drive uphill transport of a substrate. The crystal structure recently obtained for vSGLT strongly suggests that SGLT1 adopts the inverted repeat fold of the LeuT structural family for which several crystal structures are now available. What is largely missing is an accurate view of the rates at which SGLT1 transits between its different conformational states. In the present study, we used simulated annealing to analyze a large set of steady-state and pre–steady-state currents measured for human SGLT1 at different membrane potentials, and in the presence of different Na⁺ and α-methyl-d-glucose (αMG) concentrations. The simplest kinetic model that could accurately reproduce the time course of the measured currents (down to the 2 ms time range) is a seven-state model (C₁ to C₇) where the binding of the two Na⁺ ions (C₄→C₅) is highly cooperative. In the forward direction (Na⁺/glucose influx), the model is characterized by two slow, electroneutral conformational changes (59 and 100 s⁻¹) which represent reorientation of the free and of the fully loaded carrier between inside-facing and outside-facing conformations. From the inward-facing (C₁) to the outward-facing Na-bound configuration (C₅), 1.3 negative elementary charges are moved outward. Although extracellular glucose binding (C₅→C₆) is electroneutral, the next step (C₆→C₇) carries 0.7 positive charges inside the cell. Alignment of the seven-state model with a generalized model suggested by the structural data of the LeuT fold family suggests that electrogenic steps are associated with the movement of the so-called thin gates on each side of the substrate binding site. To our knowledge, this is the first model that can quantitatively describe the behavior of SGLT1 down to the 2 ms time domain. The model is highly symmetrical and in good agreement with the structural information obtained from the LeuT structural family.     

Identification of the Third Na Site and the Sequence of Extracellular Binding Events in the Glutamate Transporter

The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na⁺ ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt_ph. Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Glt_ph (the apo state, as well as in the presence of Na⁺, the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na⁺ ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na⁺ ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp³¹², Thr⁹², and Asn³¹⁰, along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na⁺ ions, reveals a stable structure—showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na⁺ ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events.     

Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters

The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na⁺-dependent reuptake of released neurotransmitters. Previous studies suggested that Na⁺-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na⁺ binding and transport (i.e. replacing Na⁺ with Li⁺ or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na⁺ cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na⁺ dependence. Thus, the detailed AIN generated from our method is shown to connect Na⁺ binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na⁺ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.     

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na⁺ and Cl^?-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na⁺-binding sites and a putative Cl^?-binding site. Mutation of all residues in the two proposed Na⁺-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na⁺-binding site 1) and mutants S20A, S20G, S20G/G405S (Na⁺-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na⁺-binding site 1 caused a decrease of the Na⁺-K_0.5, whereas mutations of site 2 increased the K_0.5. Mutation of Na⁺-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates ³⁶Cl^? demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na⁺/1Cl^?-proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.     

Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP

The Na⁺-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na⁺-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.     

Microseconds Simulations Reveal a New Sodium-binding Site and the Mechanism of Sodium-coupled Substrate Uptake by LeuT

The bacterial sodium-coupled leucine/alanine transporter LeuT is broadly used as a model system for studying the transport mechanism of neurotransmitters because of its structural and functional homology to mammalian transporters such as serotonin, dopamine, or norepinephrine transporters, and because of the resolution of its structure in different states. Although the binding sites (S1 for substrate, and Na1 and Na2 for two co-transported sodium ions) have been resolved, we still lack a mechanistic understanding of coupled Na⁺- and substrate-binding events. We present here results from extensive (>20 μs) unbiased molecular dynamics simulations generated using the latest computing technology. Simulations show that sodium binds initially the Na1 site, but not Na2, and, consistently, sodium unbinding/escape to the extracellular (EC) region first takes place at Na2, succeeded by Na1. Na2 diffusion back to the EC medium requires prior dissociation of substrate from S1. Significantly, Na⁺ binding (and unbinding) consistently involves a transient binding to a newly discovered site, Na1″, near S1, as an intermediate state. A robust sequence of substrate uptake events coupled to sodium bindings and translocations between those sites assisted by hydration emerges from the simulations: (i) bindings of a first Na⁺ to Na1″, translocation to Na1, a second Na⁺ to vacated Na1″ and then to Na2, and substrate to S1; (ii) rotation of Phe²⁵³ aromatic group to seclude the substrate from the EC region; and (iii) concerted tilting of TM1b and TM6a toward TM3 and TM8 to close the EC vestibule.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of Glt_Ph, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described Glt_Ph as an aspartate transporter driven by Na⁺ or a glutamate transporter driven by either Na⁺ or H⁺. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the Glt_Ph transport. We confirm that Glt_Ph is a Na⁺-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na⁺ ions. In contrast to the EAATs, transport via Glt_Ph is independent of H⁺ and K⁺. We propose a kinetic model of transport in which at least two Na⁺ ions are coupled to the cotransport of each aspartate molecule by Glt_Ph, and where an ion- and substrate-free transporter reorients to complete the transport cycle.     

Conformational rearrangements to the intracellular open states of the LeuT and ApcT transporters are modulated by common mechanisms

Recent crystallographic studies revealed that five transporter families without much sequence similarities among them have similar structure folds to LeuT, a bacterial neurotransmitter:sodium symporter homolog. The LeuT fold is characterized by an internal twofold structural pseudosymmetry. The transport cycle of some members of each of these families is dependent on a sodium gradient across the membrane, whereas in some others the role of sodium is mimicked by proton. We report on the identification of common structure-dynamics elements of the transporters with LeuT fold, which are recognizable in the conformational transitions related to function. The findings from comparative computational modeling and simulation studies of LeuT, and ApcT from the amino acid-polyamine-organocation transporter family define the intramolecular mechanisms by which Na⁺ binding couples to the transport process, and single out the lead/active role of TM1a in the transition to inward-open conformation. These mechanistic insights are derived in the context of collaborative investigations of LeuT dynamics with both single-molecule fluorescence and simulations that have produced excellent agreement of the dynamic details, and are found to be generalizable across the transporter families and to transcend sequence and motif similarities.     

Metadynamics Simulations Reveal a Na+ Independent Exiting Path of Galactose for the Inward-Facing Conformation of vSGLT

Sodium-Galactose Transporter (SGLT) is a secondary active symporter which accumulates sugars into cells by using the electrochemical gradient of Na⁺ across the membrane. Previous computational studies provided insights into the release process of the two ligands (galactose and sodium ion) into the cytoplasm from the inward-facing conformation of Vibrio parahaemolyticus sodium/galactose transporter (vSGLT). Several aspects of the transport mechanism of this symporter remain to be clarified: (i) a detailed kinetic and thermodynamic characterization of the exit path of the two ligands is still lacking; (ii) contradictory conclusions have been drawn concerning the gating role of Y263; (iii) the role of Na⁺ in modulating the release path of galactose is not clear. In this work, we use bias-exchange metadynamics simulations to characterize the free energy profile of the galactose and Na⁺ release processes toward the intracellular side. Surprisingly, we find that the exit of Na⁺ and galactose is non-concerted as the cooperativity between the two ligands is associated to a transition that is not rate limiting. The dissociation barriers are of the order of 11–12 kcal/mol for both the ion and the substrate, in line with kinetic information concerning this type of transporters. On the basis of these results we propose a branched six-state alternating access mechanism, which may be shared also by other members of the LeuT-fold transporters.