2.A.22 The Neurotransmitter:Sodium Symporter (NSS) Family

Members of the NSS family catalyze uptake of a variety of neurotransmitters, amino acids, osmolytes and related nitrogenous substances by a solute:Na+ symport mechanism (Rudnick et al. 2013). Sometimes Cl- is cotransported, and some exhibit a K+ dependency. The human dopamine transporter probably co-transports the positively charged or zwitterionic dopamine species with 2Na+ and 1Cl-. The human betaine/GABA transporter cotransports 3Na+ and 1 or 2Cl- with one molecule of betaine or GABA. Two different glycine transporters, GlyT1 (TC #2.A.22.2.2) and GlyT2 (TC #2.A.22.2.6), cotransport glycine with 2Na+ and 3Na+, respectively as well as 1Cl-. Most characterized members are from animals, but bacterial and archaeal homologues have been sequenced, and one bacterial homologue, TnaT of Symbiobacterium thermophilum, TC #2.A.22.5.2, has been shown to be a Na+-dependent tryptophan uptake permease with high affinity (145 nM) (Androutsellis-Theotokis et al., 2003) while a second is a tyrosine-specific Na+ symporter. Eukaryotic NSS proteins are generally of 600-800 amino acyl residues in length and possess 12 putative transmembrane helical spanners, but about 70% of prokaryotic homologues have 11 TMSs (Quick et al., 2006).  Several NSS family members have been characterized from marine invertebrates (Kinjo et al. 2013).

Neurotransmitter: sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. In eukaryotic NSS, chloride is transported together with the neurotransmitter. However, transport by the bacterial homologues LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions. Zomot et al, (2007) found that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (γ-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux (but not of exchange) was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) similarly resulted in chloride-independent transport. The reciprocal mutations in LeuT and Tyt1 rendered substrate binding and/or uptake by these bacterial NSS chloride dependent. Their data indicated that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.

Evidence supports the conclusion that some members of the NSS family are dimers while others are monomers, and still others can be oligomeric depending on their localization. Thus, the glycine transporter is monomeric in the plasma membrane but oligomeric when intracellular. Both the serotonin and dopamine transporters may be dimeric. In the latter case, the extracellular end of TMS6 may be at a symmetrical dimer interface (Hastrup et al., 2001).  In mammals, several isoforms of these transporters (e.g., DAT and NET) can be generated by tissue-specific alternative splicing (Sogawa et al. 2010).

Tavoulari et al. (2011) described conversion of the Cl- -independent prokaryotic tryptophan transporter TnaT (2.A.22.4.1) to a fully functional Cl- -dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in a serotonin transporter (SERT; 2.A.22.1.1) provided direct evidence for the involvement of each of the proposed residues in Cl- coordination. In both SERT and TnaT-D268S, Cl- and Na+ mutually increase each other's potency, consistent with am electrostatic interaction through adjacent binding sites.

Several members of the NSS family have been shown to exhibit channel-like properties under certain experimental conditions. Thus, sizable unitary ionic currents have been reported for membrane patches containing either the γ-aminobutyrate, norepinephrine or serotonin transporter. In the presence of Zn2+ (10 μM), the dopamine transporter (DAT) catalyzes uncoupled Cl- conductance (Meinild et al., 2004). Channel-like currents have also been measured for mammalian Na+/H+/K+-coupled glutamate transporters of the DAACS family (TC #2.A.23). Evidence shows that these channels can accommodate neurotransmitters as well as inorganic ions. One of these, CAATCH1 (TC #2.A.22.2.4) can function as an amino acid-gated cation (K+ and Na+) channel (Quick and Stevens, 2001). Different amino acids (pro, thr, met) differentially affect the state probability of the channel. These observations suggest that, as has been demonstrated for carriers of a few other families, neurotransmitter transporters can be induced to function as voltage-gated channels.

The GABA transporter, GAT-1 (TC #2.A.23.3.2), can catalyze channel-like fluxes of Li+ and K+. Mutations in TMS1 can lock the permease in the 'cation leak' mode (Kanner, 2003). The leak in the G63C (but not the G63S) mutant could be blocked by addition of membrane impermeable sulfhydryl reagents, suggesting that this position is accessible from the external aqueous medium. Thus, TMS1 contains determinants of both Na+-coupled GABA transport and the cation leak.

Cocaine and related drugs act by inhibiting clearance of released monoamine neurotransmitters from the synaptic cleft. Cocaine inhibits uptake of serotonin via SERT, dopamine via DAT, and norepinephrine via NET. Cocaine binds with high affinity to all three transporters, exhibiting competitive inhibition with the monoamine substrates, probably by binding to the active sites (Rasmussen et al., 2001).

The differential expression patterns and physiological roles of the glycine transporter subtypes have been exploited in the development of novel transport inhibitors to treat schizophrenia (GLYT1 inhibitors). GLYT1 transports glycine and also the N-methyl derivative of glycine, sarcosine, whereas GLYT2 only transports glycine. Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts as a coagonist with L-glutamate on the N-methyl-D-aspartate subtypes of glutamate receptors. Glycine transporters regulate glycine concentrations within both inhibitory and excitatory synapses. The GLYT1 subtypes of glycine transporters are expressed in glial cells surrounding both excitatory and inhibitory synapses, whereas the GLYT2 subtypes of glycine transporters are expressed in presynaptic inhibitory glycinergic neurons (Vandenberg et al. 2007).

There are two Na+/Cl- -dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations, and these transporters show differences in substrate selectivity and blocker sensitivity. Differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2 (Vandenberg et al., 2007).

The crystal structure of a bacterial member of the NSS family has been determined complexed to leucine and 2 Na+ (Yamashita et al., 2005). The protein core consists of the first ten of the 12 TMSs with segments 1-5 and 6-10 exhibiting a pseudo-2-fold axis in the plane of the membrane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The binding pocket of LeuT contains two metal binding sites (Caplan et al., 2008). The first ion in site NA1 is directly coupled to the bound substrte (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Double ion occupancy of the binding pocket is required to ensure substrate coupling to Na+ (but not to Li+ or K+ cations). The 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 (Caplan et al., 2008).

Substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein (Zhao et al., 2011). In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine, alanine-induced dynamics are induced in the intracellular gate region of LeuT that directly correlate with transport efficiency. Thus, binding of a second substrate (S2) in the extracellular vestibule appears to act cooperatively with the primary substrate (S1) to control intracellular gating more than 30 Å away.

In the presence of Na+, the leucine-bound state of the invertebrate neutral amino acid transporter, KAAT1 of Manduca sexta (TC#2.A.22.2.5) is supposed to be relatively stable, while in the presence of K+, and at negative potentials, the progression of the leucine-bound form along the cycle is favoured. In this context, serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity (Miszner et al., 2007). This lepidopteran neutral amino acid transporter has an unusual cation selectivity, being activated by K+ and Li+ in addition to Na+. Asp338 is essential for KAAT1 activation by K+ and for the coupling of amino acid transport to ion fluxes. Lys102 is likely to interact with Asp338 (Castagna et al., 2007). Asp338 corresponds to Asn286, a residue located in the Na+ binding site in the crystal structure of the NSS transporter LeuT. Lys102, interacting with Asp338, could contribute to the spatial organization of the KAAT1 cation binding site and the permeation pathway.

The generalized transport reaction for the members of this family is:

solute (out) + Na+ (out) → solute (in) + Na+ (in)



This family belongs to the APC Superfamily.

 

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McCoy, K.E., X. Zhou, and P.D. Vize. (2008). Collectrin/tmem27 is expressed at high levels in all segments of the developing Xenopus pronephric nephron and in the Wolffian duct. Gene Expr Patterns 8: 271-274.

Meinild, A.-K., H.H. Sitte, and U. Gether. (2004). Zinc potentiates an uncoupled anion conductance associated with the dopamine transporter. J. Biol. Chem. 279: 49671-49679.

Miszner, A., A. Peres, M. Castagna, S. Bettè, S. Giovannardi, F. Cherubino, and E. Bossi. (2007). Structural and functional basis of amino acid specificity in the invertebrate cotransporter KAAT1. J. Physiol. 581: 899-913.

Müller, H.K., O. Wiborg, and J. Haase. (2006). Subcellular redistribution of the serotonin transporter by secretory carrier membrane protein 2. J. Biol. Chem. 281: 28901-28909.

Nakanishi, T., Y. Fukuyama, M. Fujita, Y. Shirasaka, and I. Tamai. (2011). Carnitine Precursor γ-Butyrobetaine is a Novel Substrate of the Na+- and Cl--dependent GABA Transporter Gat2. Drug Metab Pharmacokinet 26: 632-636.

Noskov, S.Y., and B. Roux (2008). Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms. J. Mol. Biol. 377: 804-818.

Pedersen AV., Andreassen TF. and Loland CJ. (2014). A conserved salt bridge between transmembrane segments 1 and 10 constitutes an extracellular gate in the dopamine transporter. J Biol Chem. 289(50):35003-14.

Perez, C. and C. Ziegler. (2013). Mechanistic aspects of sodium-binding sites in LeuT-like fold symporters. Biol Chem 394: 641-648.

Quick, M. and B.R. Stevens. (2001). Amino acid transporter CAATCH1 is also an amino acid-gated cation channel. J. Biol. Chem. 276: 33413-33418.

Quick, M., H. Yano, N.R. Goldberg, L. Duan, T. Beuming, L. Shi, H. Weinstein, and J.A. Javitch. (2006). State-dependent conformations of the translocation pathway in the tyrosine transporter Tyt1, a novel neurotransmitter:sodium symporter from Fusobacterium nucleatum. J. Biol. Chem. 281: 26444-26454.

Ramamoorthy, S. and R.D. Blakely. (1999). Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants. Science 285: 763-766.

Rappold, P.M., M. Cui, A.S. Chesser, J. Tibbett, J.C. Grima, L. Duan, N. Sen, J.A. Javitch, and K. Tieu. (2011). Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3. Proc. Natl. Acad. Sci. USA 108: 20766-20771.

Rasmussen, S.G.F., F.I. Carroll, M.J. Maresch, A.D. Jensen, C.G. Tate, and U. Gether. (2001). Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter. J. Biol. Chem. 276: 4717-4723.

Reizer, J., A. Reizer, and M.H. Saier, Jr. (1994). A functional superfamily of sodium/solute symporters. Biochim. Biophys. Acta 1197: 133-166.

Rimoldi, S., E. Bossi, S. Harpaz, A.G. Cattaneo, G. Bernardini, M. Saroglia, and G. Terova. (2015). Intestinal B(0)AT1 (SLC6A19) and PEPT1 (SLC15A1) mRNA levels in European sea bass (Dicentrarchus labrax) reared in fresh water and fed fish and plant protein sources. J Nutr Sci 4: e21.

Rudnick G., Kramer R., Blakely RD., Murphy DL. and Verrey F. (2014). The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction. Pflugers Arch. 466(1):25-42.

Sahai, M.A., C. Davidson, G. Khelashvili, V. Barrese, N. Dutta, H. Weinstein, and J. Opacka-Juffry. (2016). Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances - The case of the benzofuran 5-MAPB. Prog Neuropsychopharmacol Biol Psychiatry. [Epub: Ahead of Print]

Santarelli, S., K.V. Wagner, C. Labermaier, A. Uribe, C. Dournes, G. Balsevich, J. Hartmann, M. Masana, F. Holsboer, A. Chen, M.B. Müller, and M.V. Schmidt. (2015). SLC6A15, a novel stress vulnerability candidate, modulates anxiety and depressive-like behavior: involvement of the glutamatergic system. Stress 1-8. [Epub: Ahead of Print]

Schlessinger, A., E. Geier, H. Fan, J.J. Irwin, B.K. Shoichet, K.M. Giacomini, and A. Sali. (2011). Structure-based discovery of prescription drugs that interact with the norepinephrine transporter, NET. Proc. Natl. Acad. Sci. USA 108: 15810-15815.

Schwartz, J.W., G. Novarino, D.W. Piston, and L.J. DeFelice. (2005). Substrate binding stoichiometry and kinetics of the norepinephrine transporter. J. Biol. Chem. 280: 19177-19184.

Scruggs, S.M., S. Disatian, and E.C. Orton. (2010). Serotonin transmembrane transporter is down-regulated in late-stage canine degenerative mitral valve disease. J Vet Cardiol 12: 163-169.

Sealover, N.R., B. Felts, C.P. Kuntz, R.E. Jarrard, G.H. Hockerman, E.L. Barker, and L.K. Henry. (2016). The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux. Biochem Pharmacol 120: 46-55.

Seyer, P., F. Vandermoere, E. Cassier, J. Bockaert, and P. Marin. (2016). Physical and functional interactions between the serotonin transporter and the neutral amino acid transporter ASCT2. Biochem. J. 473: 1953-1965.

Singer, D., S.M. Camargo, T. Ramadan, M. Schäfer, L. Mariotta, B. Herzog, K. Huggel, D. Wolfer, S. Werner, J.M. Penninger, and F. Verrey. (2012). Defective intestinal amino acid absorption in Ace2 null mice. Am. J. Physiol. Gastrointest Liver Physiol 303: G686-695.

Sloan, J. and S. Mager. (1999). Cloning and functional expression of a human Na+ and Cl--dependent neutral and cationic amino acid transporter B0+. J. Biol. Chem. 274: 23740-23745.

Sogawa, C., C. Mitsuhata, K. Kumagai-Morioka, N. Sogawa, K. Ohyama, K. Morita, K. Kozai, T. Dohi, and S. Kitayama. (2010). Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. PLoS One 5: e11945.

Sohail, A., K. Jayaraman, S. Venkatesan, K. Gotfryd, M. Daerr, U. Gether, C.J. Loland, K.T. Wanner, M. Freissmuth, H.H. Sitte, W. Sandtner, and T. Stockner. (2016). The Environment Shapes the Inner Vestibule of LeuT. PLoS Comput Biol 12: e1005197.

Stolzenberg, S., Z. Li, M. Quick, L. Malinauskaite, P. Nissen, H. Weinstein, J.A. Javitch, and L. Shi. (2017). The Role of TM5 in Na2 Release and the Conformational Transition of Neurotransmitter:Sodium Symporters toward the Inward-Open State. J. Biol. Chem. [Epub: Ahead of Print]

Sucic, S. and L.J. Bryan-Lluka. (2007). Investigation of the functional roles of the MELAL and GQXXRXG motifs of the human noradrenaline transporter using cysteine mutants. Eur J Pharmacol 556: 27-35.

Supplisson, S. and M.J. Roux. (2002). Why glycine transporters have different stoichiometries. FEBS Lett. 529: 93-101.

Sweeney, C.G., B.P. Tremblay, T. Stockner, H.H. Sitte, and H.E. Melikian. (2016). Dopamine Transporter Amino- and Carboxy-Termini Synergistically Contribute to Substrate and Inhibitor Affinities. J. Biol. Chem. [Epub: Ahead of Print]

Takanaga, H., B. Mackenzie, Y. Suzuki, and M.A. Hediger. (2005). Identification of mammalian proline transporter SIT1 (SLC6A20) with characteristics of classical system imino. J. Biol. Chem. 280: 8974-8984.

Tavoulari, S., A.N. Rizwan, L.R. Forrest, and G. Rudnick. (2011). Reconstructing a chloride-binding site in a bacterial neurotransmitter transporter homologue. J. Biol. Chem. 286: 2834-2842.

Tavoulari, S., E. Margheritis, A. Nagarajan, D.C. DeWitt, Y.W. Zhang, E. Rosado, S. Ravera, E. Rhoades, L.R. Forrest, and G. Rudnick. (2015). Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog. J. Biol. Chem. [Epub: Ahead of Print]

Tomi, M., A. Tajima, M. Tachikawa, and K. Hosoya. (2008). Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells. Biochim. Biophys. Acta. 1778: 2138-2142.

Trotschel C., Follmann M., Nettekoven JA., Mohrbach T., Forrest LR., Burkovski A., Marin K. and Kramer R. (2008). Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family. Biochemistry. 47(48):12698-709.

Uchiyama, T., T. Fujita, H.J. Gukasyan, K.J. Kim, Z. Borok, E.D. Crandall, and V.H. Lee. (2008). Functional characterization and cloning of amino acid transporter B(0,+) (ATB0,+) in primary cultured rat pneumocytes. J. Cell. Physiol. 214: 645-654.

Vandenberg, R.J., K. Shaddick, and P. Ju. (2007). Molecular Basis for Substrate Discrimination by Glycine Transporters. J. Biol. Chem. 282: 14447-14453.

Vincenti, S., M. Castagna, A. Peres, and V.F. Sacchi. (2000). Substrate selectivity and pH dependence of KAAT1 expressed in Xenopus laevis oocytes. J. Membr. Biol. 174: 213-224.

Walline, C.C., D.E. Nichols, F.I. Carroll, and E.L. Barker. (2008). Comparative molecular field analysis using selectivity fields reveals residues in the third transmembrane helix of the serotonin transporter associated with substrate and antagonist recognition. J Pharmacol Exp Ther 325: 791-800.

Wang, H., A. Goehring, K.H. Wang, A. Penmatsa, R. Ressler, and E. Gouaux. (2013). Structural basis for action by diverse antidepressants on biogenic amine transporters. Nature 503: 141-145.

Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., and Gouaux, E. (2005). Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437: 215-223.

Zaia, K.A. and R.J. Reimer. (2009). Synaptic Vesicle Protein NTT4/XT1 (SLC6A17) Catalyzes Na+-coupled Neutral Amino Acid Transport. J. Biol. Chem. 284: 8439-8448.

Zapata A., B. Kivell, Y. Han, J.A. Javitch, E.A. Bolan, D. Kuraguntla, V. Jaligam, M. Oz, L.D. Jayanthi, D.J. Samuvel, S. Ramamoorthy, T.S. Shippenberg. (2007). Regulation of dopamine transporter function and cell surface expression by D3 dopamine receptors. J. Biol. Chem. 282: 35842-35854.

Zhang, Y.W. and G. Rudnick. (2006). The cytoplasmic substrate permeation pathway of serotonin transporter. J. Biol. Chem. 281: 36213-36220.

Zhang, Y.W., B.E. Turk, and G. Rudnick. (2016). Control of serotonin transporter phosphorylation by conformational state. Proc. Natl. Acad. Sci. USA 113: E2776-2783.

Zhang, Y.W., J. Gesmonde, S. Ramamoorthy, and G. Rudnick. (2007). Serotonin transporter phosphorylation by cGMP-dependent protein kinase is altered by a mutation associated with obsessive compulsive disorder. J. Neurosci. 27: 10878-10886.

Zhao, C. and S.Y. Noskov. (2013). The molecular mechanism of ion-dependent gating in secondary transporters. PLoS Comput Biol 9: e1003296.

Zhao, Y., D.S. Terry, L. Shi, M. Quick, H. Weinstein, S.C. Blanchard, and J.A. Javitch. (2011). Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474: 109-113.

Zhou, Y., E. Zomot, and B.I. Kanner. (2006). Identification of a lithium interaction site in the γ-aminobutyric acid (GABA) transporter GAT-1. J. Biol. Chem. 281: 22092-22099.

Zomot, E., A. Bendahan, M. Quick, Y. Zhao, J.A. Javitch, and B.I. Kanner (2007). Mechanism of chloride interaction with neurotransmitter:sodium symporters. Nature 449: 726-730.

Examples:

TC#NameOrganismal TypeExample
2.A.22.1.1

Serotonin (5-hydroxytryptamine; 5 HT):Na+:Cl- symporter, SERT.A  Also transports amphetamines; blocked by cocaine and tricyclic antidepressants such as Prozac; interacts directly with the secretory carrier-associated membrane protein-2 (SCAMP2; O15127) to regulate the subcellular distribution (Muller et al., 2006). Uses an alternating sites mechanism with all 3 substrates bound (Zhang and Rudnick, 2006).  Molecular determinants for antidepressants in the human serotonin and norepinephrine  transporters have been identified (Andersen et al., 2011). A conserved asparagine residue in transmembrane segment 1 (TMS1) of the serotonin transporter dictates chloride-coupled neurotransmitter transport (Henry et al., 2011). The formation and breakage of ionic interactions with amino acids in transmembrane helices 6 and 8 and intracellular loop 1 may be of importance for substrate translocation (Gabrielsen et al., 2012). Methylation of the SLC6A4 gene promoter controls depression in men by an epigenetic mechanism (Devlin et al., 2010).  The 5HT Km is 0.4 micromolar (Banovic et al. 2010).  Regulated allosterically by ATM7 which stabilizes the outward-facing conformation of SERT (Kortagere et al. 2013).  Functional and regulatory mechanisms involving the N- and C-terminal hydrophilic domains have been considered (Fenollar-Ferrer et al. 2014).  The range of substrates bound and transported has been predicted (Kaufmann et al. 2009).  TMS3 may function in substrate and antagonist recognition (Walline et al. 2008).  The 3-d x-ray structure with antidepressants bound have been solved, leading to mechanistic predictions; antidepressants lock SERT in an outward- open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding (Coleman et al. 2016).  Na+ and cocaine stabilize outward-open conformations of SERT and decrease phosphorylation while agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increase phosphorylation. The opposing effects of the inhibitors, cocaine and ibogaine, were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na+ and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively (Zhang et al. 2016).  SERT is regulated by multiple molecular mechanisms including its physical interaction with intracellular proteins including the ASCT2 (alanine-serine-cysteine-threonine 2; TC# 2.A.23.3.2), co-expressed with SERT in serotonergic neurons and involved in the transport of small neutral amino acids across the plasma membrane (Seyer et al. 2016).  Transports substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) (Sealover et al. 2016).  A naturally occurring mutation, I425V, associated with obsessive-compulsive disorder and other neuropsychiatric disorders, activates hSERT and eliminates stimulation via the cyclicGMP-dependent pathway (Zhang et al. 2007).  The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. In this transporter, Glu394 plays a role in MDMA recognition (Sealover et al. 2016). Intestinal dysbiosis may upregulate SERT expression and contribute to the development of chronic constipation (Cao et al. 2017).

Animals

SERT or SLC6A4 of Homo sapiens

 
2.A.22.1.10

Serotonin transporter, Mod-5, of 671 aas and 12 TMSs.  Functions in thermotaxis memory behavior (Li et al. 2013).

Mod-5 of Caenorhabditis elegans

 
2.A.22.1.2

Noradrenaline:Na+ symporter (NET) (also transports 1-methyl-4-tetrahydropyridinium and amphetamines; it is a target of cocaine and amphetamines as well as of therapetics for depression, obsessive-compulsive disorders, and post-traumatic stress disorder. This homooligomeric transporter binds one substrate molecule per transporter subunit (Schwartz et al., 2005; Schlessinger et al., 2011; Andersen et al., 2011). Extracellular loop 3 contributes to substrate and inhibitor selectivity (Lynagh et al. 2013). The highly conserved MELAL and GQXXRXG motifs, located in the second transmembrane domain and the first intracellular loop of hNET, respectively, are determinants of NET cell surface expression, and substrate and inhibitor binding (Sucic and Bryan-Lluka 2007).

Animals

SLC6A2 of Homo sapiens

 
2.A.22.1.3

Dopamine:Na+ symporter, DAT (also takes up amphetamines in symport with Na+ which promotes intracellular Na+-dependent dopamine efflux (Khoshbouei et al., 2003)) [Inhibited by cocaine, amphetamines, neurotoxins, antidepressants and ethanol (Chen et al., 2004)]. (Zn2+ potentiates uncoupled Cl- conductance (Meinild et al., 2004)).  A conserved salt bridge between TMSs 1 and 10 constitutes an extracellular gate (Pedersen et al. 2014).  DAT is regulated by D3 dopamine receptors (Zapata et al., 2007). P25α (tubulin polymerization-promoting protein, TPPP; 094811) increases dopamine transporter localization to the plasma membrane (Fjorback et al., 2011). Mediates paraquat (an herbicide) neurotoxicity (Rappold et al., 2011).  Membrane cholesterol modulates the outward facing conformation and alters cocaine binding (Hong and Amara 2010).  Threonine 53 phosphorylation in the rat orthologue (P23977) (Serine 53 in the human transporter) regulates substrate reuptake and amphetamine-stimulated efflux (Foster et al. 2012).  DAT is enriched in filopodia and induces filopodia formation (Caltagarone et al. 2015).  Dasotraline is a novel inhibitor of dopamine and norepinephrine reuptake used for the treatment of attention-deficit/hyperactivity disorder (ADHD) (Hopkins et al. 2015). When in complex with 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB), a psychoactive adictive agonists, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT (Sahai et al. 2016).  The cytoplasmic N- and C-terminal domains contribute to substrate and inhibitor binding (Sweeney et al. 2016). DAT can exist as a monomer or a cooperative dimer subject to allosteric regulation (Cheng et al. 2017). Cocaine binds in the S1 site to stabilize an inactive form of DAT (Krout et al. 2017).

Animals

SLC6A3 of Homo sapiens

 
2.A.22.1.4Antidepressant- and cocaine-sensitive dopamine transporter, T23G5.5 (Km for dopamine, 1.2 µM; dependent on extracellular Na+ and Cl-; blocked by cocaine and D-amphetamine) (Jayanthi et al. 1998) (interacts with syntaxin 1A to regulate channel activity and dopaminergic synaptic transmission; Carvelli et al., 2008)

Animals

T23G5.5 of Caenorhabditis elegans (Q03614)

 
2.A.22.1.5High affinity octopamine transporter, OAT (also transports tyramine and dopamine in the 0.4-3.0 μM range (Donly et al., 2007)). animals (insects)OAT of Trichoplusia ni (Q95VZ4)
 
2.A.22.1.6

The dopamine/norepinephrine transporter (SmDAT) (Larsen et al. 2011).

Trematodes

DAT of Schistosoma mansoni (E9LD23)

 
2.A.22.1.7

Dopamine transporter.  The 3-d structure is known to 3.0 Å resolution (Penmatsa et al. 2013).  The crystal structure, bound to the tricyclic antidepressant nortriptyline, shows the transporter locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7.

Animals

Dopamine transporter of Drosophila melanogaster

 
2.A.22.1.8

Snf-10 transporter.  Required for protease-mediated activation of sperm motility.  Present in the plasma membrane before activation, but assumes a polarized localization to the cell body region that is dependent on membrane fusions mediated by the dysferlin FER-1 (Fenker et al. 2014).

Animals

Snf-10 of Caenorabditis elegans

 
2.A.22.1.9

The sodium-dependent serotonin transporter of 622 aas and 12 TMSs, SerT.  Terminates the action of serotonin by its high affinity reuptake into presynaptic terminals (Demchyshyn et al. 1994). Substrates have been predicted based on modeling studies (Kaufmann et al. 2009).

SerT of Drosophila melanogaster (Fruit fly)

 
Examples:

TC#NameOrganismal TypeExample
2.A.22.2.1Proline:Na+ symporter Animals Proline transporter of Rattus norvegicus
 
2.A.22.2.10

Sodium- and chloride-dependent glycine transporter 2 (GlyT-2) (GlyT2) (Solute carrier family 6 member 5).  The STAS domain has been solved by x-ray crystalography (PDB# 3LLO).  Functions to remove and recycle synaptic glycine from inhibitory synapses.  Mutations in GlyT are a common cause of hyperakplexia or startle disease in humans. The ER chaparone, calnexin, facilitates GlyT processing (Arribas-González et al. 2013).

Animals

SLC6A5 of Homo sapiens

 
2.A.22.2.11Sodium-dependent proline transporter (Solute carrier family 6 member 7)AnimalsSLC6A7 of Homo sapiens
 
2.A.22.2.12Sodium- and chloride-dependent glycine transporter 1 (GlyT-1) (GlyT1) (Solute carrier family 6 member 9)AnimalsSLC6A9 of Homo sapiens
 
2.A.22.2.13

Sodium-dependent nutrient amino acid transporter 1 (DmNAAT1)

AnimalsNAAT1 of Drosophila melanogaster
 
2.A.22.2.2Glycine:Na+ symporter, GlyT1c (glycine/2Na+/1Cl- symporter)Animals Glycine transporter (GlyT1c) of Rattus norvegicus
 
2.A.22.2.3

Neutral and cationic amino acid:Na+:Cl- symporter, B0+. The rat homologue (NP_001032633) transports basic and zwitterionic amino acids, but not proline, aspartic acid and glutamic acid (Uchiyama et al, 2008).

Animals

SLC6A14 of Homo sapiens

 
2.A.22.2.4

Gut epithelium absorptive neutral amino acid Na+- or K+-dependent transporter, CAATCH1 (electrogenic; Cl--independent. Substrates: L-proline-preferring + Na+; L-threonine-preferring + K+; also transports L-methionine) (CAATCH1 can also function as an amino acid-gated cation [Na+ and K+] channel.)

Animals

Neutral amino acid transporter CAATCH1 of Manduca sexta

 
2.A.22.2.5Gut epithelium absorptive neutral amino acid, K+- and Na+-dependent transporter KAAT1 (electrogenic; Cl--dependent; activated by alkaline pH; all zwiterionic amino acids except methyl AIB are substrates). CAATCH1 is 95% identical to KAAT1. Leu > Thr and Pro.AnimalsNeutral amino acid transporter KAAT1 of Manduca sexta
 
2.A.22.2.6

Glycine:Na+ transporter, GlyT2b (glycine/3Na+/1Cl- symporter, SLC6A5). GlyT2 and VIAAT cooperate to determine the vesicular glycinergic phenotype (Aubrey et al., 2007). Startle disease in Irish wolfhounds is associated with a microdeletion in the glycine transporter GlyT2 gene (Gill et al., 2011). A dominant hyperekplexia (startle disease) mutation Y705C in humans alters trafficking and the biochemical properties of GlyT2 (Gimenez et al. 2012).

Animals

Glycine transporter (GlyT2b) of Mus musculus

 
2.A.22.2.7

Acetylcholine/choline:Na+ symporter, Snf-6 (interacts with dystrophin which determines its localization to the neuromuscular junction) (Kim et al., 2004)

Animals

Snf-6 of Caenorhabditis elegans (O76689)

 
2.A.22.2.8Cation-dependent nutrient amino acid transporter, AAT1 (L-phe > cys > his > ala > ser > met > ile > tyr > D-phe > thr > gly) (Bondko et al., 2005)Animals

AAT1 of Aedes aegypti (Q6VS78)

 
2.A.22.2.9

The Densovirus type-2 (BmDNV-2) receptor; putative amino acid transporter (625aas;11-12TMSs)

Animals

Nsd-2 of Bombyx mori (B2ZXL8)

 
Examples:

TC#NameOrganismal TypeExample
2.A.22.3.1

Betaine/GABA:Na+ symporter, BGT1. (Substrates include: betaine, GABA, diaminobutyrate, β-alanine, proline, quinidine, dimethylglycine, glycine, and sarcosine with decreasing affinity in that order).  Selective inhibitors have been identified (Kragholm et al. 2013).

Animals

SLC6A12 of Homo sapiens

 
2.A.22.3.10Sodium- and chloride-dependent GABA transporter 2 (GAT-2) (Solute carrier family 6 member 13)AnimalsSLC6A13 of Homo sapiens
 
2.A.22.3.11Sodium- and chloride-dependent creatine transporter 1 (CT1) (Creatine transporter 1) (Solute carrier family 6 member 8)AnimalsSLC6A8 of Homo sapiens
 
2.A.22.3.12

Sodium- and chloride-dependent GABA transporter, Ine (Protein inebriated) (Protein receptor oscillation A)

AnimalsIne of Drosophila melanogaster
 
2.A.22.3.2

γ-aminobutyric acid (GABA):Na+:Cl- symporter, GAT-1 (Stoichiometry, GABA:Na+ = 1:2 where both Na+ binding sites, Na1 and Na2, have been identified. Na2 but not Na1 can accommodate Li+ (Zhou et al., 2006)). Glutamine 291 is essential for Cl- binding (Ben-Yona et al., 2011). Four human isoforms have been identified, GAT-1, GAT-2, GAT-3, and GAT-4, all about 70% identical to each other (Borden et al., 1992). GAT-2 transports γ-aminobutyric acid and β-alanine (Christiansen et al, 2007) It also concentratively takes up β-alanine and α-fluoro-β-alanine (Liu et al., 1999). GAT1 is capable of intracellular Na+-, Cl-- and GABA-induced outward currents (reverse GABA transport; GABA efflux) (Bertram et al., 2011). An acidic amino acid residue in transmembrane helix 10 conserved in the Neurotransmitter:Sodium:Symporters is essential for the formation of the extracellular gate of GAT-1 (Ben-Yona and Kanner, 2012). It is required for stringent gating and tight coupling of ion- and substrate-fluxes in the GABA transporter family (Dayan et al. 2017). GAT-1 is the target of the antiepileptic drug, tiagabine (Kardos et al. 2010). The monomeric protein has been purified fused to GFP (Hu et al. 2017).

Animals

SLC6A1 of Homo sapiens

 
2.A.22.3.3

The taurine:Na+ symporter, TauT or SLC6A6 (also transports β-alanine and γ-aminobutyric acid (GABA); Tomi et al., 2008; Anderson et al., 2009).

AnimalsSLC6A6 of Homo sapiens
 
2.A.22.3.4Creatine:Na+ symporter Animals Creatine transporter of Oryctolagus cuniculus
 
2.A.22.3.5Renal apical membrane creatine:Na2+:Cl- symporter (CRT) (Garcia-Delgado et al., 2007)Animals CRT of Rattus norvegicus (P28570)
 
2.A.22.3.6γ-aminobutyric acid (GABA):Na+:Cl- symporter GAT-1 (stoichiometry = 1:2:1) (Jiang et al., 2005)AnimalsGAT-1 of Caenorhabditis elegans (AAT02634)
 
2.A.22.3.7The GABA transporter, GAT4 (single mutations render this transporter C1- independent) (Zomot et al., 2007)AnimalsGABA transporter GAT4 of Mus musculus (Q8BWA7)
 
2.A.22.3.8

Mouse GABA, β-alanine, fluoro-β-alanine and taurine transporter-3 (GAT3) (Liu et al. 1999). Orthologous to rat and human GAT2; 72% identical to GAT4 (2.A.22.3.7) (takes up GABA with high affinity into presynaptic terminals). Also takes up the carnitine precursor, gamma-butyrobetaine (Nakanishi et al., 2011).

Animals

GAT3 of Mus musculus (P31649)

 
2.A.22.3.9Sodium- and chloride-dependent GABA transporter 3 (GAT-3) (Solute carrier family 6 member 11)AnimalsSLC6A11 of Homo sapiens
 
Examples:

TC#NameOrganismal TypeExample
2.A.22.4.1High affinity tryptophan:Na+ symporter, TnaT (Androutsellis-Theotokis et al., 2003)Bacteria TnaT of Symbiobacterium thermophilum
 
2.A.22.4.2

The amino acid (leucine):2 Na+ symporter, LeuTAa (Yamashita et al., 2005). LeuT possesses two ion binding sites, NA1 and NA2, both highly specific for Na+ but with differing mechanisms of binding (Noskov and Roux, 2008). X-ray structures have been determined for LeuT in substrate-free outward-open and apo inward-open states (Krishnamurthy and Gouaux, 2012).  Extracytoplasmic substrate binding at an allosteric site controls activity (Zhao et al. 2011).  It has been proposed that the 5 TMS repeat derived from a DedA domain (9.B.27; Khafizov et al. 2010).  Mechanistic aspect of Na+ binding have been studied (Perez and Ziegler 2013).  Structural studies of mutant LeuT proteins suggest how antidepressants bind to biogenic amine transporters (Wang et al. 2013).  The detailed mechanism was studied by Zhao and Noskov, 2013.  Uptake involves movement of the substrate amino acid from the outward facing binding site, S1, to the inward facing binding site, S2, coupled with confrmational changes in the protein (Cheng and Bahar 2013).  The complete substrate translocation pathway has been proposed (Cheng and Bahar 2014). The inward facing conformation of LeuT has been solved (Grouleff et al. 2015).  Substrate-induced unlocking of the inner gatemay determinethe catalytic efficiency of the transporter (Billesbølle et al. 2015). Of the two Na+ binding sites, occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na+ and transmembrane helices 1 and 8 whereas Na+ binding at Na1 influences conformational change through a network of intermediary interactions (Tavoulari et al. 2015). TMS1A movements revealed a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure, especiallly with respect to the inner vestibule (Sohail et al. 2016).

Bacteria

LeuTAa of Aquifex aeolicus (2A65_A)

 
2.A.22.4.3The methionine/alanine uptake porter, MetPS (Trotschel et al., 2008) (MetP is the transporter; MetS is an essential auxiliary subunit).

Bacteria

MetPS of Corynebacterium glutamicum
MetP (563aas; Q8NRL8)
MetS (60aas; Q8NRL9)

 
Examples:

TC#NameOrganismal TypeExample
2.A.22.5.1Hypothetical Na+-dependent permease Archaea MJ1319 of Methanococcus jannaschii
 
2.A.22.5.2The 11 TMS Na+-dependent tyrosine transporter, Tyt1 (Quick et al., 2006)BacteriaTyt1 of Fusobacterium nucleatum (Q8RHM5)
 
2.A.22.5.3

Neurotransmitter:sodium symporter of 455 aas, MhsT.  The x-ray structures of two occluded inward-facing states with bound Na+ ions and L-tryptophan have been solved (4US4; Malinauskaite et al. 2014).  These structures provide insight into the cytoplasmic release of Na+. The switch from outward- to inward-oriented states is centered on the partial unwinding of transmembrane helix 5, facilitated by a conserved GlyX9Pro motif that opens an intracellular pathway for water to access the Na+2 site. Solvation through this TMS 5 pathway may facilitate Na+ release from the Na+2 site to the inward-open state (Malinauskaite et al. 2014). TMS5 plays a role in the binding and release of Na+ from the Na+2 site and in mediating conformational changes (Stolzenberg et al. 2017).

Firmicutes

MhsT of Bacillus halodurans

 
2.A.22.5.4

Uncharacterized protein of 427 aas and 12 TMSs.

UP of Thermococcus profundus

 
Examples:

TC#NameOrganismal TypeExample
2.A.22.6.1

Na+/Amino acid transporter 1, SIT1/IMINO (SLC6A20). Transports imino acids such as proline (Km=0.2 mM), pipecolate, and N-methylated amino acids such as MeAIB and sarcosine (Na+-dependent, Cl--stimulated, pH-independent, voltage-dependent) (Li+, but not H+ can substitute for Na+) (Takanaga et al., 2005). It is a 2Na+/1Cl--proline cotransporter (Bröer et al., 2009).

Animals

SIT1 of Rattus norvegicus (Q64093)

 
2.A.22.6.2

Synaptic vesicle neutral amino acid:Na+ symporter NTT4/XT1/BOAT3 (SLC6A17) (catalyzes uptake of neurotransmitters into presynaptic vesicles (Zaia and Reimer, 2009).

Animals

NTT4 of Rattus norvegicus (P31662)

 
2.A.22.6.3

Kidney and intestinal apical membrane epithelial transporter for Na+-dependent, Cl--independent reabsorption of neutral amino acids. Many neutral L-amino acids bind with ~0.5 mM affinities Leu is the preferred substrate, but all large neutral non-aromatic L-amino acids bind to this transporter. Uptake of leucine is sodium-dependent. In contrast to other members of the neurotransmitter transporter family, this one does not appear to be chloride-dependent.  Activity is enhanced by collectrin (Tmem27), a collecting duct transmembrane (1 TMS) glycoprotein (Q9HBJ8) (Danilczyk et al., 2006). The Hartnup Disorder protein (mouse orthologue, (Q9D687) (Broer et al., 2004; 2008) forms a complex with collectrin and the brush border carboxypeptidase angiotensin-converting enzyme 2 (ACE2). Mutation as in Hartnup disorder (B0AT1(R240Q)) decreases complex formation and leads to neutral aminoaciduria and in some cases pellagra-like symptoms (Kowalczuk et al., 2008; Singer et al. 2012).  Collectrin is expressed in the simple embryonic kidney of amphibians such as Xenopus, the pronephros, at high levels (McCoy et al. 2008).           

Animals

SLC6A19 of Homo sapiens

 
2.A.22.6.4

The neutral amino acid transporter, B0AT3 (Slc6a18); XT2 (55% identical to 2.A.22.6.3)

AnimalsSLC6A18 of Homo sapiens
 
2.A.22.6.5 solute carrier family 6, member 16AnimalsSLC6A16 of Homo sapiens
 
2.A.22.6.6

Sodium-dependent vesicular neutral amino acid transporter SLC6A17 (Sodium-dependent neurotransmitter transporter NTT4/BOAT3) (Solute carrier family 6 member 17) (Hägglund et al. 2013).

Animals

SLC6A17 of Homo sapiens

 
2.A.22.6.7

Sodium-dependent neutral amino acid transporter B(0)AT2 (Sodium- and chloride-dependent neurotransmitter transporter NTT73) (Sodium-coupled branched-chain amino-acid transporter 1) (Solute carrier family 6 member 15) (Transporter v7-3).  It is mainly expressed in neurons and plays a role in depression and stress vulnerability (Santarelli et al. 2015).

Animals

SLC6A15 of Homo sapiens

 
2.A.22.6.8

Sodium- and chloride-dependent transporter XTRP3 (Sodium/amino-acid transporter 1) (Solute carrier family 6 member 20) (Transporter rB21A homologue)

Animals

SLC6A20 of Homo sapiens

 
2.A.22.6.9

Sea bass amino acid uptake porter, SLC6A19 or B0AT1 of 634 aas.  Levels depend on diet (Rimoldi et al. 2015).

Animals

SLC6A19 of Dicentrarchus labrax (European seabass) (Morone labrax)