Elucidating the Mechanism Behind Sodium-Coupled Neurotransmitter Transporters by Reconstitution.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today's single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters.

Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.

SLC6 transporter oligomerization.

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

How structural elements evolving from bacterial to human SLC6 transporters enabled new functional properties.

BACKGROUND: Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters are involved in an expanded set of functional properties to the eukaryotic transporters. These functional properties are not shared by the bacterial homologs, which lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters that have been suggested to involve these structural elements are sparse or merely descriptive. RESULTS: To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov state model analysis of experimentally determined properties of the wild-type and mutant hDAT constructs. These offer a quantitative outline of mechanisms in which a rich spectrum of interactions of the hDAT N-terminus and C-terminus contribute to the regulation of transporter function (e.g., by phosphorylation) and/or to entirely new phenotypes (e.g., reverse uptake (efflux)) that were added in evolution. CONCLUSIONS: The findings are consistent with the proposal that the size of eukaryotic neurotransmitter transporter termini increased during evolution to enable more functions (e.g., efflux) not shared with the bacterial homologs. The mechanistic explanations for the experimental findings about the modulation of function in DAT, the serotonin transporter, and other eukaryotic transporters reveal separate roles for the distal and proximal segments of the much larger N-terminus in eukaryotic transporters compared to the bacterial ones. The involvement of the proximal and distal segments - such as the role of the proximal segment in sustaining transport in phosphatidylinositol 4,5-bisphosphate-depleted membranes and of the distal segment in modulating efflux - may represent an evolutionary adaptation required for the function of eukaryotic transporters expressed in various cell types of the same organism that differ in the lipid composition and protein complement of their membrane environment.

An unfolding story: Small molecules remedy misfolded monoamine transporters.

The key role of monoamine transporters is to take up neurotransmitters from the synaptic cleft and rapidly terminate neurotransmission. Monoamine transporters begin their journey by folding in the endoplasmic reticulum. Upon achieving their natively-folded state, the oligomerized transporters engage the coat protein complex II machinery and exit the endoplasmic reticulum compartment in a concentrative fashion. The transporters are subsequently sorted in the endoplasmic reticulum-Golgi intermediate complex and the Golgi apparatus, prior to reaching their pivotal site of action at the plasma membrane. Stringent quality-control mechanisms ensure that only the correctly-folded protein cargo departs the endoplasmic reticulum. Genetic point mutations in the coding sequences of monoamine transporters can trigger severe physiologic deficiencies by inducing folding defects. Protein misfolding precludes the delivery of functional monoamine transporters to the cell surface. Chemical- and/or pharmacological-chaperone molecules, which facilitate folding, have proven effective in restoring the activity of several misfolded pathological variants of monoamine transporters.

The Environment Shapes the Inner Vestibule of LeuT.

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.

Conformational Dynamics on the Extracellular Side of LeuT Controlled by Na+ and K+ Ions and the Protonation State of Glu290.

Ions play key mechanistic roles in the gating dynamics of neurotransmitter:sodium symporters (NSSs). In recent microsecond scale molecular dynamics simulations of a complete model of the dopamine transporter, a NSS protein, we observed a partitioning of K(+) ions from the intracellular side toward the unoccupied Na2 site of dopamine transporter following the release of the Na2-bound Na(+) Here we evaluate with computational simulations and experimental measurements of ion affinities under corresponding conditions, the consequences of K(+) binding in the Na2 site of LeuT, a bacterial homolog of NSS, when both Na(+) ions and substrate have left, and the transporter prepares for a new cycle. We compare the results with the consequences of binding Na(+) in the same apo system. Analysis of >50-µs atomistic molecular dynamics and enhanced sampling trajectories of constructs with Glu(290), either charged or neutral, point to the Glu(290) protonation state as a main determinant in the structural reconfiguration of the extracellular vestibule of LeuT in which a "water gate" opens through coordinated motions of residues Leu(25), Tyr(108), and Phe(253) The resulting water channel enables the binding/dissociation of the Na(+) and K(+) ions that are prevalent, respectively, in the extracellular and intracellular environments.

Molecular dynamics simulations of Na(+) and leucine transport by LeuT.

Molecular dynamics simulations are used to gain insight into the binding of Na(+) and leucine substrate to the bacterial amino acid transporter LeuT, focusing on the crystal structures of LeuT in the outward-open and inward-open states. For both conformations of LeuT, a third Na(+) binding site involving Glu290 in addition to the two sites identified from the crystal structures is observed. Once the negative charge from Glu290 in the inward-open LeuT is removed, the ion bound to the third site is ejected from LeuT rapidly, suggesting that the protonation state of Glu290 regulates Na(+) binding and release. In Cl(-)-dependent transporters where Glu290 is replaced by a neutral serine, a Cl(-) ion would be required to replace the role of Glu290. Thus, the simulations provide insights into understanding Na(+) and substrate transport as well as Cl(-)-independence of LeuT.

Functional mechanisms of neurotransmitter transporters regulated by lipid-protein interactions of their terminal loops.

The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP(2)) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP(2) lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP(2) lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins. This article is part of a Special Issue entitled: Lipid-protein interactions.

Cooperativity between individual transporter protomers: new data fuelling old complexes.

Neurotransmitter transporters are arranged in an oligomeric quaternary structure as evidenced by crosslinking or fluorescence resonance energy transfer (FRET)-microscopy. In a study by Zhen and colleagues highlighted by this Editorial in the current issue of Journal of Neurochemistry, the combination of mutant and wild-type dopamine transporter (DAT) has been used to establish the cooperation between transporter protomers; the DAT mutant version has an altered affinity for the radiolabelled inhibitor [³H]CFT. Zhen and colleagues predict how saturation-binding curves ought to look, if the two binding sites (i.e. of the wild type and the mutant DAT) operated independently. The results are clear-cut: the experimental observations are inconsistent with curves obtained by mixing independent binding sites. Thus, by definition, the binding sites cooperate. Read the full article 'Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities' on page 167.

Accelerated molecular dynamics and protein conformational change: a theoretical and practical guide using a membrane embedded model neurotransmitter transporter.

Molecular dynamics simulation provides a powerful and accurate method to model protein conformational change, yet timescale limitations often prevent direct assessment of the kinetic properties of interest. A large number of molecular dynamic steps are necessary for rare events to occur, which allow a system to overcome energy barriers and conformationally transition from one potential energy minimum to another. For many proteins, the energy landscape is further complicated by a multitude of potential energy wells, each separated by high free-energy barriers and each potentially representative of a functionally important protein conformation. To overcome these obstacles, accelerated molecular dynamics utilizes a robust bias potential function to simulate the transition between different potential energy minima. This straightforward approach more efficiently samples conformational space in comparison to classical molecular dynamics simulation, does not require advanced knowledge of the potential energy landscape and converges to the proper canonical distribution. Here, we review the theory behind accelerated molecular dynamics and discuss the approach in the context of modeling protein conformational change. As a practical example, we provide a detailed, step-by-step explanation of how to perform an accelerated molecular dynamics simulation using a model neurotransmitter transporter embedded in a lipid cell membrane. Changes in protein conformation of relevance to the substrate transport cycle are then examined using principle component analysis.

Using a collection of MUPP1 domains to investigate the similarities of neurotransmitter transporters C-terminal PDZ motifs.

A ubiquitous feature of neurotransmitter transporters is the presence of short C-terminal PDZ binding motifs acting as important trafficking elements. Depending on their very C-terminal sequences, PDZ binding motifs are usually divided into at least three groups; however this classification has recently been questioned. To introduce a 3D aspect into transporter's PDZ motif similarities, we compared their interactions with the natural collection of all 13 PDZ domains of the largest PDZ binding protein MUPP1. The GABA, glycine and serotonin transporters showed unique binding preferences scattered over one or several MUPP1 domains. On the contrary, the dopamine and norepinephrine transporter PDZ motifs did not show any significant affinity to MUPP1 domains. Interestingly, despite their terminal sequence diversity all three GABA transporter PDZ motifs interacted with MUPP1 domain 7. These results indicate that similarities in binding schemes of individual transporter groups might exist. Results also suggest the existence of variable PDZ binding modes, allowing several transporters to interact with identical PDZ domains and potentially share interaction partners in vivo.

Conformational dynamics of ligand-dependent alternating access in LeuT.

The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na(+)- and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.

How LeuT shapes our understanding of the mechanisms of sodium-coupled neurotransmitter transporters.

Neurotransmitter transporters are ion-coupled symporters that drive the uptake of neurotransmitters from neural synapses. In the past decade, the structure of a bacterial amino acid transporter, leucine transporter (LeuT), has given valuable insights into the understanding of architecture and mechanism of mammalian neurotransmitter transporters. Different conformations of LeuT, including a substrate-free state, inward-open state, and competitive and non-competitive inhibitor-bound states, have revealed a mechanistic framework for the transport and transport inhibition of neurotransmitters. The current review integrates our understanding of the mechanistic and pharmacological properties of eukaryotic neurotransmitter transporters obtained through structural snapshots of LeuT.

Neurotransmitter transporters: structure meets function.

At synapses, sodium-coupled transporters remove released neurotransmitters, thereby recycling them and maintaining a low extracellular concentration of the neurotransmitter. The molecular mechanism underlying sodium-coupled neurotransmitter uptake is not completely understood. Several structures of homologs of human neurotransmitter transporters have been solved with X-ray crystallography. These crystal structures have spurred a plethora of computational and experimental work to elucidate the molecular mechanism underlying sodium-coupled transport. Here, we compare the structures of GltPh, a glutamate transporter homolog, and LeuT, a homolog of neurotransmitter transporters for the biogenic amines and inhibitory molecules GABA and glycine. We relate these structures to data obtained from experiments and computational simulations, to draw conclusions about the mechanism of uptake by sodium-coupled neurotransmitter transporters. Here, we propose how sodium and substrate binding is coupled and how binding of sodium and substrate opens and closes the gates in these transporters, thereby leading to an efficient coupled transport.

Labeling of neuronal receptors and transporters with quantum dots.

The ability to efficiently visualize protein targets in cells is a fundamental goal in biological research. Recently, quantum dots (QDots) have emerged as a powerful class of fluorescent probes for labeling membrane proteins in living cells because of breakthrough advances in QDot surface chemistry and biofunctionalization strategies. This review discusses the increasing use of QDots for fluorescence imaging of neuronal receptors and transporters. The readers are briefly introduced to QDot structure, photophysical properties, and common synthetic routes toward the generation of water-soluble QDots. The following section highlights several reports of QDot application that seek to unravel molecular aspects of neuronal receptor and transporter regulation and trafficking. This article is closed with a prospectus of the future of derivatized QDots in neurobiological and pharmacological research.

Monoamine transporter structure, function, dynamics, and drug discovery: a computational perspective.

With the breakthrough crystallization of the bacterial leucine transporter protein LeuT, the first available X-ray structure for the neurotransmitter/sodium symporter family, development of 3-D computational models is suddenly essential for structure-function studies on the plasmalemmal monoamine transporters (MATs). LeuT-based MAT models have been used to guide elucidation of substrate and inhibitor binding pockets, and molecular dynamics simulations using these models are providing insight into conformations involved in the substrate translocation cycle. With credible MAT models finally in hand, structure-based virtual screening for novel ligands is yielding lead compounds toward the development of new medications for psychostimulant dependence, attention deficit hyperactivity, depression, anxiety, schizophrenia, and other disorders associated with dopamine, norepinephrine, or serotonin dysregulation.

Templates and models of monoamine transporter proteins.

X-ray crystallography, structural bioinformatics and computational chemistry have become important techniques in the discovery and development of effective and safe new drugs. From a drug discovery point of view, membrane proteins are among the most interesting molecular targets, but the current knowledge about detailed 3D structures of membrane proteins is sparse. Homology modeling techniques may provide structural knowledge about membrane proteins and their interactions with drugs and other molecules. The neurotransmitter sodium symporters (NSS) are the molecular targets of many pharmacologically active substances, and we have used three different secondary transporters as templates for modeling the NSS proteins DAT, NET and SERT. The first template was based on the electron density projection map of the Escherichia coli Na+/H+ antiporter (NhaA), while later the X-ray structure of Lac Permease (symporter) was used as a template. The helical architectures of these templates have a lot in common, and models based on both could contribute with structural explanations of several experimental studies in spite of low homology with NSS proteins. In 2005 the crystal structure of a bacterial homologue of the human monoamine neurotransmitter transporter Aquifex aeolicus (LeuTAa) was reported. This structure was the first experimental structure of a NSS family member, and represented a breakthrough for homology modeling of pharmacological important NSS proteins. Since then several X-ray structures LeuTAa in complex with pharmacologically important compounds have been published. Homology models of NSS proteins, combined with site-directed mutagenesis data, have identified ligand binding sites and contributed with important knowledge for new drug development.

Cytoplasmic permeation pathway of neurotransmitter transporters.

Ion-coupled solute transporters are responsible for transporting nutrients, ions, and signaling molecules across a variety of biological membranes. Recent high-resolution crystal structures of several transporters from protein families that were previously thought to be unrelated show common structural features indicating a large structural family representing transporters from all kingdoms of life. This review describes studies that led to an understanding of the conformational changes required for solute transport in this family. The first structure in this family showed the bacterial amino acid transporter LeuT, which is homologous to neurotransmitter transporters, in an extracellularly oriented conformation with a molecule of leucine occluded at the substrate site. Studies with the mammalian serotonin transporter identified positions, buried in the LeuT structure, that defined a potential pathway leading from the cytoplasm to the substrate binding site. Modeling studies utilized an inverted structural repeat within the LeuT crystal structure to predict the conformation of LeuT in which the cytoplasmic permeation pathway, consisting of positions identified in SERT, was open for diffusion of the substrate to the cytoplasm. From the difference between the model and the crystal structures, a simple "rocking bundle" mechanism was proposed, in which a four-helix bundle changed its orientation with respect to the rest of the protein to close the extracellular pathway and open the cytoplasmic one. Subsequent crystal structures from structurally related proteins provide evidence supporting this model for transport.