The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na⁺, Cl⁻, and K⁺ gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na⁺-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca²⁺ (but not other cations) to functionally replace Na⁺ for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca²⁺ and Na⁺ illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca²⁺ promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na⁺-binding sites.     

4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter

Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.     

Helix XI contributes to the entrance of the serotonin transporter permeation pathway

The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP(+) transport. We examined the importance of TMH XI residues F551 and F556 for MPP(+) translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP(+) transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP(+) transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP(+) was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuT(Aa)) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway.     

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.     

Ionic currents in the human serotonin transporter reveal inconsistencies in the alternating access hypothesis

We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore.     

Regulation of glial glutamate transporters by C-terminal domains

Excitatory amino acid transporter 2 (EAAT2) is a high affinity glutamate transporter predominantly expressed in astroglia. Human EAAT2 encompasses eight transmembrane domains and a 74-amino acid C-terminal domain that resides in the cytoplasm. We examined the role of this region by studying various C-terminal truncations and mutations using heterologous expression in mammalian cells, whole-cell patch clamp recording and confocal imaging. Removal of the complete C terminus (K498X EAAT2) results in loss of function because of intracellular retention of truncated proteins in the cytoplasm. However, a short stretch of amino acids (E500X EAAT2) within the C terminus results in correctly processed transporters. E500X reduced glutamate transport currents by 90%. Moreover, the voltage and substrate dependence of E500X EAAT2 anion currents was significantly altered. WT and mutant EAAT2 anion channels are modified by external Na(+) in the presence as well as in the absence of L-glutamate. Whereas Na(+) stimulates EAAT2 anion currents in the presence of L-glutamate, increased [Na(+)] reduces such currents without glutamate. In cells internally dialyzed with Na(+), WT, and truncated EAAT2 display comparable Na(+) dependence. With K(+) as main internal cation, E500X drastically increased the apparent dissociation constant for external Na(+). The effects of E500X can be represented by a kinetic model that allows translocation of the empty transporter from the outward- to the inward-facing conformation and stabilization of the inward-facing conformation by internal K(+). Our results demonstrate that the C terminus modifies the glutamate uptake cycle, possibly affecting the movements of the translocation domain of EAAT2 glutamate transporter.     

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter’s engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.     

Glutamate and monoamine transporters: new visions of form and function

Neurotransmitters are rapidly removed from the extracellular space primarily through the actions of plasma membrane transporters. This uptake process is not only essential in the termination of neurotransmission but also serves to replenish intracellular levels of transmitter for further release. Neurotransmitter transporters couple the inward movement of substrate to the movement of Na(+) down a concentration gradient and, in addition to their transport function, some carriers also display channel-like activities. Five Na(+)/K(+)-dependent glutamate transporter subtypes belong to the solute carrier 1 (SLC1) family and a second family, SLC6, encompasses the Na(+)/Cl(-)-dependent transporters for dopamine, 5-hydroxytryptamine (serotonin), noradrenaline, GABA and glycine. Recent advances, including high-resolution structures from both families, are now providing new insights into the molecular determinants that contribute to substrate translocation and ion channel activities. Other influential studies have explored how cellular regulatory mechanisms modulate transporter function, and how the different functions of the carrier shape the patterns of neurotransmitter signaling. This review focuses on recent studies of glutamate and monoamine transporters as prototypes of the two carrier families.     

Comparison of computational model and X-ray crystal structure of human serotonin transporter: potential application for the pharmacology of human monoamine transporters

Abstract

The human serotonin transporter (hSERT) played a significant role in neurological process whose structural basis had been analysed for many years. Recently, the first homology model was constructed for hSERT based on the crystal structure of drosophila melanogaster dopamine transporter was published, and the inhibitory mechanism underlying the binding mode between hSERT and approved antidepressants was substantially investigated by molecular dynamics (MD) simulation. Right after this publication, the X-ray crystallographic structures of hSERT were reported, which provided a good opportunity to reassess the performance of previous simulation. In this study, the analyses of side-chain contact map, stereochemical quality and ligand-binding pocket were firstly conducted, which revealed that the constructed homology model of hSERT could successfully reproduce the reported crystal structure. Secondly, the approved antidepressant escitalopram was docked into the X-ray structure, and its binding pose was consistent with the reported docking pose in the homology model. Finally, MD simulation were performed based on the crystal structure of hSERT, and structural features revealed as critical for escitalopram-hSERT interaction by previous simulation were successfully recaptured. Thus, the newly reported X-ray crystal structure of hSERT was precisely predicted by computational model, which demonstrated its reliability in understanding the pharmacology of other human monoamine transporters whose 3-D structure remained unknown.     

The mechanism of substrate release by the aspartate transporter GltPh: insights from simulations

Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, Glt(Ph), has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of Glt(Ph). In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na(+) to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate-protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na(+), the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.     

The role of local hydration and hydrogen-bonding dynamics in ion and solute release from ion-coupled secondary transporters

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.     

Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.     

Structural dynamics of the monoamine transporter homolog LeuT from accelerated conformational sampling and channel analysis

The bacterial leucine transporter LeuT retains significant secondary structure similarities to the human monoamine transporters (MAT) such as the dopamine and serotonin reuptake proteins. The primary method of computational study of the MATs has been through the use of the crystallized LeuT structure. Different conformations of LeuT can give insight into mechanistic details of the MAT family. A conformational sampling performed through accelerated molecular dynamics simulations testing different combinations of the leucine substrate and bound sodium ions revealed seven distinct conformational clusters. Further analysis has been performed to target salt‐bridge residues R30–D404, Y108–F253, and R5–D369 and transmembrane domains on both the seven isolated structures and the total trajectories. In addition, solvent accessibility of LeuT and its substrate binding pockets has been analyzed using a program for calculating channel radii. Occupation of the Na2 site stabilizes the outward conformation and should bind to the open outward conformation before the leucine and Na1 sodium while two possible pathways were found to be available for intracellular transport. Proteins 2014; 82:2289–2302. © 2014 Wiley Periodicals, Inc.     

Binding and transport in norepinephrine transporters. Real-time,spatially resolved analysis in single cells using a fluorescent substrate

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP(+) (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP(+) accumulation is Na(+)-, Cl(-)-, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP(+) competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na(+) and Cl(-), binding is independent of Na(+) and actually increases in low Cl(-). We further demonstrate that ASP(+) interacts with transporters not only in transfected cells but in cultured neurons. ASP(+) is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.     

A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction.

Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl- dependent serotonin uptake, with estimated Km and Vmax values of 436 +/- 19 nm and 3.8 +/- 0.6 x 10-18 mol.cell.min-1, respectively. The Manduca high affinity Na+/Cl- dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 micro m) and cocaine (IC50 = 12.89 micro m). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148-152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 micro m). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1-146/MasSERT106-587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 micro m) compared to the human (IC50 = 0.431 micro m) and Manduca serotonin transporters. The chimera MasSERT1-67/hSERT109-630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 micro m) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine.     

Structural insights into ABC transporter mechanism

ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.     

Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter

Na+/Cl--dependent neurotransmitter transporters form constitutive oligomers. The topological arrangement is not known, but a leucine heptad repeat in transmembrane domain (TM) 2 and a glycophorin-like motif in TM6 have been proposed to stabilize the oligomer. To determine the topology, we generated versions of the human serotonin transporter (hSERT) that carried cyan or yellow fluorescent proteins at their amino and/or carboxyl terminus. Appropriate pairs were coexpressed to measure fluorescence resonance energy transfer (FRET). Donor photobleaching FRET microscopy was employed to deduce the following arrangement: within the monomer, the amino and carboxyl termini are in close vicinity. In addition, in the oligomer, the carboxyl termini are closer to each other than the amino termini. Hence, a separate interaction domain (i.e. distinct from TM2 and TM6) must reside in the carboxyl-terminal half of hSERT. This was confirmed by expressing the amino- and carboxyl-terminal halves of hSERT. These were retained intracellularly; they also retained the coexpressed full-length transporter by forming export-deficient oligomers and, when cotransfected in all possible combinations, supported FRET. Hence, both the carboxyl and amino termini contain elements that drive oligomerization. By employing fragments comprising two neighboring TM helices, we unequivocally identified TM11/12 as a new contact site by donor photobleaching FRET and beta-lactamase protein fragment complementation assay. TM1/2 was also found to self-associate. Thus, oligomerization of hSERT involves at least two discontinuous interfaces. The currently identified interaction sites drive homophilic interactions. This is consistent with assembly of SERT oligomers in an array-like structure containing multimers of dimers.     

Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.     

Ion binding and internal hydration in the multidrug resistance secondary active transporter NorM investigated by molecular dynamics simulations

Recently, a 3.65 ? resolution structure of the transporter NorM from the multidrug and toxic compound extrusion family has been determined in the outward-facing conformation. This antiporter uses electrochemical gradients to drive substrate export of a large class of antibiotic and toxic compounds in exchange for small monovalent cations (H(+) and Na(+)), but the molecular details of this mechanism are still largely unknown. Here we report all-atom molecular dynamics simulations of NorM, with and without the bound Na(+) cation and at different ion concentrations. Spontaneous binding of Na(+) is observed in several independent simulations with transient ion binding to D36 being necessary to reach the final binding site for which two competitive binding modes occur. Finally, the simulations indicate that the extracellular vestibule of the transporter invariably loses its characteristic V shape indicated by the crystallographic data, and it is reduced to a narrow permeation pathway lined by polar residues that can act as a specific pore for the transport of small cations. This event, together with the available structures of evolutionarily related transporters of the major facilitator superfamily (MFS), suggests that differences in the hydrophobic content of the extracellular vestibule may be characteristic of multidrug resistance transporters in contrast to substrate-selective members of the MFS.     

Transport mechanisms in acetylcholine and monoamine storage.

Sequence-related vesicular acetylcholine transporter (VAChT) and vesicular monoamine transporter (VMAT) transport neurotransmitter substrates into secretory vesicles. This review seeks to identify shared and differentiated aspects of the transport mechanisms. VAChT and VMAT exchange two protons per substrate molecule with very similar initial velocity kinetics and pH dependencies. However, vesicular gradients of ACh in vivo are much smaller than the driving force for uptake and vesicular gradients of monoamines, suggesting the existence of a regulatory mechanism in ACh storage not found in monoamine storage. The importance of microscopic rather than macroscopic kinetics in structure-function analysis is described. Transporter regions affecting binding or translocation of substrates, inhibitors, and protons have been found with photoaffinity labeling, chimeras, and single-site mutations. VAChT and VMAT exhibit partial structural and mechanistic homology with lactose permease, which belongs to the same sequence-defined superfamily, despite opposite directions of substrate transport. The vesicular transporters translocate the first proton using homologous aspartates in putative transmembrane domain X (ten), but they translocate the second proton using unknown residues that might not be conserved between them. Comparative analysis of the VAChT and VMAT transport mechanisms will aid understanding of regulation in neurotransmitter storage.