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A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter*

  • Anders V. Pedersen
    Affiliations
    Department of Neuroscience and Pharmacology, Molecular Neuropharmacology Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark
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  • Thorvald F. Andreassen
    Affiliations
    Department of Neuroscience and Pharmacology, Molecular Neuropharmacology Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark
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  • Claus J. Loland
    Correspondence
    To whom correspondence should be addressed: Dept. of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45-2875-6407.
    Affiliations
    Department of Neuroscience and Pharmacology, Molecular Neuropharmacology Laboratory, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark
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  • Author Footnotes
    * This work was supported in part by funds from the Danish Independent Research Council, Sapere Aude (to C. J. L.), the Lundbeck Foundation (to C. J. L.), and the UNIK Center for Synthetic Biology (to C. J. L.).
Open AccessPublished:October 22, 2014DOI:https://doi.org/10.1074/jbc.M114.586982
      Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [3H]CFT. Also, the coordination of Zn2+ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [3H]CFT binding (Bmax). Importantly, Zn2+ also inhibited [3H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [3H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.

      Introduction

      The neurotransmitter:sodium symporters (NSSs)
      The abbreviations used are: NSS
      neurotransmitter:sodium symporter
      DAT
      dopamine transporter
      dDAT
      Drosophila DAT
      synDAT
      synthetic human DAT
      SERT
      serotonin transporter
      MTSET
      [2-(trimethyl-ammonium)ethyl]-methanethiosulfonate
      TM
      transmembrane segment
      CFT
      2β-carbomethoxy-3β-(4-fluorophenyl)tropane
      S-CIT
      (S)-citalopram
      GAT-1
      GABA transporter-1
      [3H]DA
      3,4-[Ring-2,5,6-3H]-dihydroxyphenylethylamine
      UB
      uptake buffer.
      encompass a family of secondary active transporters that use the Na+ gradient across the plasma membrane as a driving force to transport solutes against their concentration gradient. Within the NSS family, we find the transporters for the monoamines dopamine (DA), norepinephrine, and serotonin. They are localized to the presynaptic terminals where they mediate rapid reuptake of the respective neurotransmitter and thereby control synaptic signaling tonus. Because of their central role in monoamine signaling, it is not surprising that the transporters are important pharmacological targets; the classical tricyclic antidepressants inhibit both the transporters for norepinephrine and serotonin (SERT), the selective serotonin reuptake inhibitors inhibit SERT, and the stimulatory effects of cocaine and amphetamine are caused by the interaction of these compounds with SERT, norepinephrine transporter, and the dopamine transporter (DAT).
      Despite their physiological and pharmacological importance, there are still many unanswered questions relating to the structural basis and molecular mechanisms behind substrate transport. Attempts to determine the tertiary structure of mammalian NSS proteins have so far proven unsuccessful mainly because of problems with obtaining sufficient amounts of purified protein of appropriate stability. These problems have been overcome by expression of either bacterial homologues to the mammalian NSS proteins, such as LeuT from Aquifex aeolicus (
      • Yamashita A.
      • Singh S.K.
      • Kawate T.
      • Jin Y.
      • Gouaux E.
      Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters.
      ), or invertebrate transporters, such as the Drosophila melanogaster DAT (dDAT) (
      • Penmatsa A.
      • Wang K.H.
      • Gouaux E.
      X-ray structure of dopamine transporter elucidates antidepressant mechanism.
      ). LeuT is a Na+-coupled transporter with specificity for the hydrophobic amino acids glycine, alanine, methionine, and leucine (
      • Yamashita A.
      • Singh S.K.
      • Kawate T.
      • Jin Y.
      • Gouaux E.
      Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters.
      ,
      • Singh S.K.
      • Piscitelli C.L.
      • Yamashita A.
      • Gouaux E.
      A competitive inhibitor traps LeuT in an open-to-out conformation.
      ). High resolution structures of LeuT have revealed a protein with 12 transmembrane segments (TMs) organized in a pseudo 2-fold symmetry axis between TM1–5 and TM6–10 with a binding site for substrate localized central in the protein (S1 site) flanked by TM1, TM3, TM6, and TM8 (
      • Yamashita A.
      • Singh S.K.
      • Kawate T.
      • Jin Y.
      • Gouaux E.
      Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters.
      ). So far, LeuT has been crystallized in three distinct conformations: outward open, outward occluded, and an inward open conformation (
      • Yamashita A.
      • Singh S.K.
      • Kawate T.
      • Jin Y.
      • Gouaux E.
      Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters.
      ,
      • Krishnamurthy H.
      • Gouaux E.
      X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.
      ). Although significant homology exists between LeuT and the mammalian NSS proteins, there are also divergent structures, including the loop domains and the much longer N and C termini found in the mammalian transporters. Despite these differences, LeuT has been used as a model protein for studying the dynamics and conformational changes that underlie substrate translocation in the mammalian NSS proteins (
      • Kazmier K.
      • Sharma S.
      • Quick M.
      • Islam S.M.
      • Roux B.
      • Weinstein H.
      • Javitch J.A.
      • McHaourab H.S.
      Conformational dynamics of ligand-dependent alternating access in LeuT.
      ,
      • Zhao Y.
      • Terry D.S.
      • Shi L.
      • Quick M.
      • Weinstein H.
      • Blanchard S.C.
      • Javitch J.A.
      Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue.
      ,
      • Claxton D.P.
      • Quick M.
      • Shi L.
      • de Carvalho F.D.
      • Weinstein H.
      • Javitch J.A.
      • McHaourab H.S.
      Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters.
      ,
      • Zhao Y.
      • Terry D.
      • Shi L.
      • Weinstein H.
      • Blanchard S.C.
      • Javitch J.A.
      Single-molecule dynamics of gating in a neurotransmitter transporter homologue.
      ). The recent crystallization of dDAT in an outward open conformation revealed a structural fold very similar to LeuT, supporting that LeuT is a valid model for eukaryotic NSS proteins at least for structural inferences (
      • Penmatsa A.
      • Wang K.H.
      • Gouaux E.
      X-ray structure of dopamine transporter elucidates antidepressant mechanism.
      ).
      It is believed that the accessibility to the central substrate binding site from either side of the membrane is controlled by the concerted movements of specific gating domains within the transporters (
      • Kniazeff J.
      • Loland C.J.
      • Goldberg N.
      • Quick M.
      • Das S.
      • Sitte H.H.
      • Javitch J.A.
      • Gether U.
      Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8.
      ,
      • Loland C.J.
      • Grånäs C.
      • Javitch J.A.
      • Gether U.
      Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding.
      ,
      • Loland C.J.
      • Norregaard L.
      • Litman T.
      • Gether U.
      Generation of an activating Zn2+ switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle.
      ,
      • Forrest L.R.
      • Zhang Y.W.
      • Jacobs M.T.
      • Gesmonde J.
      • Xie L.
      • Honig B.H.
      • Rudnick G.
      Mechanism for alternating access in neurotransmitter transporters.
      ,
      • Zhang Y.W.
      • Rudnick G.
      The cytoplasmic substrate permeation pathway of serotonin transporter.
      ,
      • Chen J.G.
      • Rudnick G.
      Permeation and gating residues in serotonin transporter.
      ,
      • Chen N.
      • Rickey J.
      • Berfield J.L.
      • Reith M.E.
      Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding.
      ,
      • Chen N.
      • Zhen J.
      • Reith M.E.
      Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine.
      ). The constellation of the gating domains should be mediated by a network of dynamic interaction between specific residues. The existence of such “gating residues” has been confirmed by inferences from the crystal structures of LeuT. In the outward occluded conformation of LeuT, access to the S1 site from the extracellular side is blocked in part by a water-mediated salt bridge formed by an arginine in TM1 (Arg-30) and an aspartate in TM10 (Asp-404). In the outward facing conformation, the Arg-30/Asp-404 salt bridge is broken, resulting in the exposure of the substrate binding site to the extracellular aqueous environment (
      • Krishnamurthy H.
      • Gouaux E.
      X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.
      ), whereas the ionic interaction is direct without a water molecule present in the inward facing conformation (
      • Yamashita A.
      • Singh S.K.
      • Kawate T.
      • Jin Y.
      • Gouaux E.
      Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters.
      ,
      • Krishnamurthy H.
      • Gouaux E.
      X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.
      ). Thus, according to the current crystal structures of LeuT, it is likely that Arg-30/Asp-404 forms a functionally important so-called “thin gate” as opposed to the “thick gate” on the intracellular side of the outward occluded conformation, which consists of 22 Å densely packed protein. Sequence alignment of the NSS members shows that the positive and negative charge in the two positions are almost completely conserved within the family (
      • Beuming T.
      • Shi L.
      • Javitch J.A.
      • Weinstein H.
      A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na+ symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function.
      ), substantiating the importance of this putative gate in the function of this class of proteins. However, to date most inferences about the role of the interaction rely on the solved crystal structures of LeuT and molecular modeling of mammalian transporters based on the LeuT (
      • Plenge P.
      • Shi L.
      • Beuming T.
      • Te J.
      • Newman A.H.
      • Weinstein H.
      • Gether U.
      • Loland C.J.
      Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter.
      ,
      • Beuming T.
      • Kniazeff J.
      • Bergmann M.L.
      • Shi L.
      • Gracia L.
      • Raniszewska K.
      • Newman A.H.
      • Javitch J.A.
      • Weinstein H.
      • Gether U.
      • Loland C.J.
      The binding sites for cocaine and dopamine in the dopamine transporter overlap.
      ,
      • Sinning S.
      • Musgaard M.
      • Jensen M.
      • Severinsen K.
      • Celik L.
      • Kolds⊘ H.
      • Meyer T.
      • Bols M.
      • Jensen H.H.
      • Schi⊘tt B.
      • Wiborg O.
      Binding and orientation of tricyclic antidepressants within the central substrate site of the human serotonin transporter.
      ); hence, whether the two residues also interact in mammalian NSS proteins and what role they have in substrate transport and inhibitor binding are yet to be established. Importantly, the role of the aspartate has been studied in the GABA transporter-1 (GAT-1), where it was shown that Asp-451, the cognate residue to Asp-404 in LeuT, only can be replaced by a glutamate if activity must be retained and that the D451E mutant shifts the conformational equilibrium of GAT-1 toward a more outward facing configuration (
      • Ben-Yona A.
      • Kanner B.I.
      An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1.
      ). Further, the same group elegantly showed that the impaired transport efficiency in D451E can be rescued by a similar mutation in a presumed intracellular gating residue (D410E), suggesting a functional connectivity between the two (
      • Ben-Yona A.
      • Kanner B.I.
      Functional defects in the external and internal thin gates of the γ-aminobutyric acid (GABA) transporter GAT-1 can compensate each other.
      ).
      In DAT, it has been proposed that, upon binding of dopamine, a hydrogen bond is formed between Asp-79 and Tyr-156 in TM3 (
      • Beuming T.
      • Kniazeff J.
      • Bergmann M.L.
      • Shi L.
      • Gracia L.
      • Raniszewska K.
      • Newman A.H.
      • Javitch J.A.
      • Weinstein H.
      • Gether U.
      • Loland C.J.
      The binding sites for cocaine and dopamine in the dopamine transporter overlap.
      ). These apparent gating residues are located right above the binding site and could cause the initial closure of the external side, but this interaction is not conserved in LeuT and has no apparent connection to the more extracellularly located Arg-85 and Asp-476 (equivalent to Arg-30 and Asp-404 in LeuT). Here, we provide experimental data demonstrating the existence in DAT of a functional interaction between the conserved residues Arg-85 in TM1 and Asp-476 in TM10 (Fig. 1). First, we substantiate the functional importance of the two residues by showing that individual charge-reversing mutations at the two loci eliminate [3H]DA uptake and dramatically decrease Bmax for binding of the cocaine analogue ((-)-2β-carbomethoxy-3β-(4-fluorophenyl)tropane ([3H]CFT). Next, we find that charge reversal at both sites partially restores [3H]CFT binding, consistent with an interaction between the two residues. In further support of a structural and functional importance of the interaction between the two residues, we show that coordination of Zn2+ between inserted histidines in the two positions potentiates [3H]CFT binding and block [3H]DA uptake. Finally, we provide evidence for the presence of the corresponding salt bridge in the homologous SERT, and we demonstrate that mutational disruption of the putative interaction renders an inserted cysteine in position 159 (I159C) in TM3 more exposed to inactivation by the sulfhydryl-reactive agent, MTSET ([2-(trimethylammonium) ethyl]methane thiosulfonate), This suggests a shift of the transporter toward a more outward open configuration and supports an important role of the TM1/TM10 interaction in regulating conformational transitions in the transport cycle.
      Figure thumbnail gr1
      FIGURE 1Presumed localization of the investigated residues in DAT. A, two-dimensional schematic representation of the human DAT. Red circles indicate the locations of the two investigated residues, Arg-85 and Asp-476, in TM1 and TM10, respectively. The open circles represent amino acid residues ordered in TMs and loop regions. The presumed glycosylation sites are indicated with small filled circles. B, molecular model of the human DAT based on the LeuT outward facing closed structure with dopamine (spheres in blue (carbons), gray (hydrogens), and red (oxygens)) docked in the center of the protein. Only a fraction of the protein is shown (gray helices and loops). The two presumed gating residues, Arg-85 and Asp-476, are depicted as orange sticks. The residues presumably closing the dopamine accessibility from the extracellular environment, Asp-79 and Tyr-165, are shown as thin black sticks. TM11 and 12 are removed for clarity.

      DISCUSSION

      The family of NSS proteins is thought to function by an alternating access mechanism, which requires the presence of gates that mediate alternated accessibility to the central substrate binding site from either the extracellular or intracellular environment. Inferences from crystal structures have provided valuable information about the likely nature of these gates, but their presence and implication in the translocation mechanism in the mammalian NSS proteins are still to be determined in detail. Here, we provide experimental data suggesting that a pair of two highly conserved residues in DAT (Arg-85 in TM1 and Asp-476 in TM10) forms a dynamic interaction that is critical for the translocation of substrate, as well as for stabilizing the binding site for the cocaine analogue, CFT.
      The charge reversal experiments show that it is possible to restore binding in the completely nonfunctional mutant, DAT R85D, by the insertion of an arginine residue (D476R) in the opposing position. The D476R per se does also perturb function; therefore in a nonrescue situation, one would expect a nonfunctional double mutant. Thus, we interpret the restoration of [3H]CFT binding in the DAT R85D/D476R mutant, with affinity comparable with WT, as a rescue situation. The absence of a complete rescue in R85D/D476R is not surprising because the swapped positions are unlikely to permit an ideal coordination of the residues relative to each other. In addition, the swapped residues will most likely form suboptimal interactions with neighboring residues, leading to potential destabilization of the protein. An example of a possible residue is the Asp-313 in TM6, which could cause suboptimal coordination of the inserted aspartate residue in the R85D/D476R. However, mutation of Asp-313 did not change the binding properties in the R85D/D476R mutant (data not shown). Of interest, we have previously performed a similar charge reversal/rescue experiment on residues constituting part of the intracellular gate in DAT (Arg-30 and Asp-436) (
      • Kniazeff J.
      • Shi L.
      • Loland C.J.
      • Javitch J.A.
      • Weinstein H.
      • Gether U.
      An intracellular interaction network regulates conformational transitions in the dopamine transporter.
      ). In this case, we were also only able to obtain a partial rescue (
      • Kniazeff J.
      • Shi L.
      • Loland C.J.
      • Javitch J.A.
      • Weinstein H.
      • Gether U.
      An intracellular interaction network regulates conformational transitions in the dopamine transporter.
      ). The partial rescue is further substantiated by the complete lack of leak currents in all mutants. It has previously been suggested that the leak current is induced by the inward facing conformation (
      • Borre L.
      • Andreassen T.F.
      • Shi L.
      • Weinstein H.
      • Gether U.
      The second sodium site in the dopamine transporter controls cation permeation and is regulated by chloride.
      ,
      • Schicker K.
      • Uzelac Z.
      • Gesmonde J.
      • Bulling S.
      • Stockner T.
      • Freissmuth M.
      • Boehm S.
      • Rudnick G.
      • Sitte H.H.
      • Sandtner W.
      Unifying concept of serotonin transporter-associated currents.
      ), suggesting that the suboptimal coordination in the R85D/D476R is not sufficient for the transporter to isomerize to the inward facing conformation and thus not mediate transport. It was, however, able to bind DA albeit with lower affinity. In this context, it is interesting to note that the interaction has to undergo several dynamic changes during the translocation cycle. According to available crystal structures of LeuT, the charged pair form a water-mediated salt bridge in the outward occluded state but a direct salt bridge in the inward facing configuration. In the outward facing conformation of both LeuT and dDAT, the residues are too far apart to interact (
      • Penmatsa A.
      • Wang K.H.
      • Gouaux E.
      X-ray structure of dopamine transporter elucidates antidepressant mechanism.
      ,
      • Krishnamurthy H.
      • Gouaux E.
      X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.
      ). Likely, the charge reversal does not support this dynamic feature. This was further substantiated by zinc site engineering. We have previously taken advantage of this approach in our study of this class of proteins where it has contributed to the elucidation of tertiary structure information (
      • Loland C.J.
      • Norregaard L.
      • Gether U.
      Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn2+-binding site.
      ,
      • Norregaard L.
      • Frederiksen D.
      • Nielsen E.O.
      • Gether U.
      Delineation of an endogenous zinc-binding site in the human dopamine transporter.
      ,
      • MacAulay N.
      • Bendahan A.
      • Loland C.J.
      • Zeuthen T.
      • Kanner B.I.
      • Gether U.
      Engineered Zn2+ switches in the γ-aminobutyric acid (GABA) transporter-1: differential effects on GABA uptake and currents.
      ,
      • Norregaard L.
      • Visiers I.
      • Loland C.J.
      • Ballesteros J.
      • Weinstein H.
      • Gether U.
      Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites.
      ) and identification of the cocaine binding site in DAT, as well as the allosteric binding site in SERT (
      • Plenge P.
      • Shi L.
      • Beuming T.
      • Te J.
      • Newman A.H.
      • Weinstein H.
      • Gether U.
      • Loland C.J.
      Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter.
      ). Here, the zinc site results not only show that the inserted His-85 and His-476 are in close proximity, but they also support the suggestion that a dynamic interplay between the residues is required for DA transport, analogous with what can deduced from the crystal structures of LeuT and dDAT (
      • Yamashita A.
      • Singh S.K.
      • Kawate T.
      • Jin Y.
      • Gouaux E.
      Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters.
      ,
      • Penmatsa A.
      • Wang K.H.
      • Gouaux E.
      X-ray structure of dopamine transporter elucidates antidepressant mechanism.
      ,
      • Krishnamurthy H.
      • Gouaux E.
      X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.
      ).
      The binding of Zn2+ to DAT R85H/H193K/D476H causes an increase in Bmax for [3H]CFT, but interestingly it apparently does not increase its affinity. This contrasts with the classical pharmacological concept that, at saturating conditions, all possible binding sites should be occupied which should result in similar Bmax but with different affinities for CFT in the two conditions. A similar effect of Zn2+ on [3H]CFT binding was observed in the DAT WT with Zn2+ binding to the endogenous site (
      • Loland C.J.
      • Norregaard L.
      • Gether U.
      Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn2+-binding site.
      ,
      • Norregaard L.
      • Frederiksen D.
      • Nielsen E.O.
      • Gether U.
      Delineation of an endogenous zinc-binding site in the human dopamine transporter.
      ). One possible explanation is that CFT alone is unable to change the conformational equilibrium of DAT toward the conformation prone to bind CFT, it is only the conformational fluctuation of the protein per se that determines whether a CFT binding site is formed. Thus, irrespective of the added CFT concentration, the DAT fraction prone to bind CFT would be the same. In contrast, Zn2+ is able to change the conformational equilibrium of DAT shifting it toward the CFT prone conformation by the coordination of R85H and D476H (or by binding to the endogenous Zn2+ binding site). Accordingly, the CFT affinity would remain the same, and the Bmax will increase. Further experiments will have to elucidate the nature of Zn2+ binding to DAT. Taken together, these Zn2+ data substantiate the close proximity between Arg-85 and Asp-476 and support the role of dynamic TM1/TM10 interactions for stabilizing inhibitor binding and promoting transport.
      In the present study, we show that the formation of the thin gate facilitates the binding of the tested ligands to DAT (CFT) and SERT (S-CIT). This is in agreement with our previous published molecular docking models of the same setup, DAT:CFT and SERT:S-CIT, based on the structure of LeuT (
      • Plenge P.
      • Shi L.
      • Beuming T.
      • Te J.
      • Newman A.H.
      • Weinstein H.
      • Gether U.
      • Loland C.J.
      Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter.
      ,
      • Beuming T.
      • Kniazeff J.
      • Bergmann M.L.
      • Shi L.
      • Gracia L.
      • Raniszewska K.
      • Newman A.H.
      • Javitch J.A.
      • Weinstein H.
      • Gether U.
      • Loland C.J.
      The binding sites for cocaine and dopamine in the dopamine transporter overlap.
      ). The interaction was also observed by other groups in molecular docking models of SERT with bound imipramine (
      • Sinning S.
      • Musgaard M.
      • Jensen M.
      • Severinsen K.
      • Celik L.
      • Kolds⊘ H.
      • Meyer T.
      • Bols M.
      • Jensen H.H.
      • Schi⊘tt B.
      • Wiborg O.
      Binding and orientation of tricyclic antidepressants within the central substrate site of the human serotonin transporter.
      ) or (S)-citalopram (
      • Andersen J.
      • Olsen L.
      • Hansen K.B.
      • Taboureau O.
      • J⊘rgensen F.S.
      • J⊘rgensen A.M.
      • Bang-Andersen B.
      • Egebjerg J.
      • Str⊘mgaard K.
      • Kristensen A.S.
      Mutational mapping and modeling of the binding site for (S)-citalopram in the human serotonin transporter.
      ). Thus, the data presented here support previously published docking models, suggesting that the binding of inhibitors induces the outward occluded conformation. This is in contrast to the published crystal structure of the dDAT with nortriptyline bound (
      • Penmatsa A.
      • Wang K.H.
      • Gouaux E.
      X-ray structure of dopamine transporter elucidates antidepressant mechanism.
      ). Here the Arg-Asp interaction is broken, suggesting that nortriptyline binds to outward open conformation. Interestingly, the same outward open conformation was shown to bind both selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, clomipramine, and the stimulant mazindol to a LeuT:biogenic amine transporter hybrid (LeuBAT) (
      • Wang H.
      • Goehring A.
      • Wang K.H.
      • Penmatsa A.
      • Ressler R.
      • Gouaux E.
      Structural basis for action by diverse antidepressants on biogenic amine transporters.
      ). Further investigations are necessary to elucidate the binding conformations induced by the different inhibitors in the mammalian monoamine transporters.
      The MTSET experiments support the hypothesis that mutation of the Arg-85/Asp-476 interaction shifts the conformational equilibrium toward a higher fraction of transporters residing in the outward open conformation. The probing for changes in conformational stages of the outer gate by investigating the MTSET reactivity toward an inserted cysteine into position 159 has become a well established method used in both DAT, the norepinephrine transporter, and SERT (
      • Loland C.J.
      • Grånäs C.
      • Javitch J.A.
      • Gether U.
      Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding.
      ,
      • Chen J.G.
      • Rudnick G.
      Permeation and gating residues in serotonin transporter.
      ,
      • Loland C.J.
      • Desai R.I.
      • Zou M.F.
      • Cao J.
      • Grundt P.
      • Gerstbrein K.
      • Sitte H.H.
      • Newman A.H.
      • Katz J.L.
      • Gether U.
      Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors.
      ). Accordingly, we show here that even relatively conserved mutations (R85H or D476N) change the susceptibility of Cys-159 for reacting with MTSET, suggesting an increased accessibility of the cysteine residue toward the extracellular aqueous environment (Fig. 5).
      Finally, we show that the shift of the charges in the corresponding residues in SERT results in a similar rescue pattern as we observed in DAT. As opposed to DAT, it is the R104E that possesses residual binding capacity in SERT. The Asp-476 in DAT is a glutamate in SERT, and mutation to arginine completely abolishes [3H]S-CIT binding (Fig. 6). Analogous to the charge reversal in DAT, the loss of binding in E493R is partly rescued by the R104E mutation. Despite the difference in binding pattern of the single mutants, the data do suggest that the two residues also interact in SERT.
      In all, we here provide evidence that the DAT residues Arg-85 and Asp-476, shown to form the thin gate in LeuT, also interact in DAT. The interaction between the two positions promotes the binding of [3H]CFT but, more importantly, the data also show that constraining the residues with Zn2+ inhibits the transport of DA, suggesting that the interaction is dynamic and must be continuously broken and formed during substrate translocation. Moreover, the presence of the charged pair throughout NSS family proteins and the presumed conserved functional role from LeuT to DAT presented here strongly supports an evolutionary conserved functional role for the two residues among all NSS proteins.

      Acknowledgments

      We thank Drs. Lei Shi and Thijs Beuming (Medical School of Cornell University, New York) for providing the molecular docking model of DAT:DA complex and Lone Rosenquist, Pia Elsman, and Bente Bennicke for excellent technical assistance. We also thank Prof. Ulrik Gether for critical reading and helpful comments to the manuscript.

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