Abstract
Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1â5 related to 6â10 by a pseudo-two-fold axis in the membrane plane. 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 structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Vanhatalo, S. & Soinila, S. The concept of chemical neurotransmission â variations on the theme. Ann. Med. 19, 151â158 (1998)
Masson, J., Sagne, C., Hamon, M. & Mestikawy, S. E. Neurotransmitter transporters in the central nervous system. Pharm. Rev. 51, 439â464 (1999)
Hahn, M. K. & Blakely, R. D. Monoamine transporter gene structure and polymorphisms in relation to psychiatric and other complex disorders. Pharmacogenomics J. 2, 217â235 (2002)
Richerson, G. B. & Wu, Y. Role of the GABA transporter in epilepsy. Adv. Exp. Med. Biol. 548, 76â91 (2004)
Amara, S. G. & Sonders, M. S. Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend. 51, 87â96 (1998)
Krogsgaard-Larsen, P., Frolund, B. & Frydenvang, K. GABA uptake inhibitors. Design, molecular pharmacology and therapeutic aspects. Curr. Pharm. Des. 6, 1193â1209 (2000)
Barker, E. L. & Blakely, R. D. in Psychopharmacologyâthe Fourth Generation of Progress (eds Bloom, F. E. & Kupfer, D. J.) (Raven Press, New York, 2000)
Guastella, J. et al. Cloning and expression of a rat brain GABA transporter. Science 249, 1303â1306 (1990)
Nelson, N. The family of Na+/Cl--dependent neurotransmitter transporters. J. Neurochem. 71, 1785â1803 (1998)
Torres, G. E., Gainetdinov, R. R. & Caron, M. G. Plasma membrane monoamine transporters: structure, regulation, and function. Nature Rev. Neurosci. 4, 13â25 (2003)
Chen, J. G., Liu-Chen, S. & Rudnick, G. Determination of external loop topology in the serotonin transporter by site-directed chemical labeling. J. Biol. Chem. 273, 12675â12681 (1998)
Bismuth, Y., Kavanaugh, M. P. & Kanner, B. I. Tyrosine 140 of the γ-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition. J. Biol. Chem. 272, 16096â16102 (1997)
Chen, J. G., Sachpatzidis, A. & Rudnick, G. The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding. J. Biol. Chem. 272, 28321â28327 (1997)
Cao, Y., Li, M., Mager, S. & Lester, H. A. Amino acid residues that control pH modulation of transport-associated current in mammalian serotonin transporters. J. Neurosci. 18, 7739â7749 (1998)
Rudnick, G. in Neurotransmitter Transporters: Structure, Function, and Regulation (ed. Reith, E. A.) 25â52 (Humana Press, Totowa, New Jersey, 2002)
Kavanaugh, M. P., Arriza, J. L., North, R. A. & Amara, S. G. Electrogenic uptake of γ-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes. J. Biol. Chem. 267, 22007â22009 (1992)
Roux, M. & Supplisson, S. Neuronal and glial glycine transporters have different stoichiometries. Neuron 25, 373â383 (2000)
Androutsellis-Theotokis, A. et al. Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters. J. Biol. Chem. 278, 12703â12709 (2003)
Hendrickson, W. A. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51â58 (1991)
Murata, K. et al. Structural determinants of water permeation through aquaporin-1. Nature 407, 599â605 (2000)
Hirai, T. et al. Three-dimensional structure of a bacterial oxalate transporter. Nature Struct. Biol. 9, 597â600 (2002)
Chen, J. G., Liu-Chen, S. & Rudnick, G. External cysteine residues in the serotonin transporter. Biochemistry 36, 1479â1486 (1997)
Wang, J. B., Moriwaki, A. & Uhl, G. R. Dopamine transporter cysteine mutants: second extracellular loop cysteines are required for transporter expression. J. Neurochem. 64, 1416â1419 (1995)
Just, H., Sitte, H. H., Schmid, J. A., Freissmuth, M. & Kudlacek, O. Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter. J. Biol. Chem. 279, 6650â6657 (2004)
Sitte, H. H., Farhan, H. & Javitch, J. A. Sodium-dependent neurotransmitter transporters: oligomerization as a determinant of transporter function and trafficking. Mol. Interv. 4, 38â47 (2004)
Ponce, J., Biton, B., Benavides, J., Avenet, P. & Aragon, C. Transmembrane domain III plays an important role in ion binding and permeation in the glycine transporter GLYT2. J. Biol. Chem. 275, 13856â13862 (2000)
Keshet, G. I. et al. Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1. FEBS Lett. 371, 39â42 (1995)
Nayal, M. & Di Cera, E. Valence screening of water in protein crystals reveals potential Na+ binding sites. J. Mol. Biol. 256, 228â234 (1996)
Harding, M. M. Metal-ligand geometry relevant to proteins and in proteins: sodium and potassium. Acta Crystallogr. D 58, 872â874 (2002)
Mager, S. et al. Ion binding and permeation at the GABA transporter GAT1. J. Neurosci. 16, 5405â5414 (1996)
Penado, K. M., Rudnick, G. & Stephan, M. M. Critical amino acid residues in transmembrane span 7 of the serotonin transporter identified by random mutagenesis. J. Biol. Chem. 273, 28098â28106 (1998)
Chen, N. & Reith, M. E. Na+ and the substrate permeation pathway in dopamine transporters. Eur. J. Pharmacol. 479, 213â221 (2003)
Mari, S. A. et al. Aspartate 338 contributes to the cationic specificity and to driver-amino acid coupling in the insect cotransporter KAAT1. Cell. Mol. Life Sci. 61, 243â256 (2004)
Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969â970 (1966)
Pantanowitz, S., Bendahan, A. & Kanner, B. I. Only one of the charged amino acids located in the transmembrane alpha-helices of the γ-aminobutyric acid transporter (subtype A) is essential for its activity. J. Biol. Chem. 268, 3222â3225 (1993)
Bennett, E. R., Su, H. & Kanner, B. I. Mutation of arginine 44 of GAT-1, a (Na+ + Cl-)-coupled γ-aminobutyric acid transporter from rat brain, impairs net flux but not exchange. J. Biol. Chem. 275, 34106â34113 (2000)
Loland, C. J., Granas, C., Javitch, J. A. & Gether, U. Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding. J. Biol. Chem. 279, 3228â3238 (2004)
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. Proc. Natl Acad. Sci. USA 99, 1683â1688 (2002)
Toyoshima, C. & Nomura, H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418, 605â611 (2002)
Lopez-Corcuera, B., Nunez, E., Martinez-Maza, R., Geerlings, A. & Aragon, C. Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2. J. Biol. Chem. 276, 43463â43470 (2001)
Sato, Y., Zhang, Y. W., Androutsellis-Theotokis, A. & Rudnick, G. Analysis of transmembrane domain 2 of rat serotonin transporter by cysteine scanning mutagenesis. J. Biol. Chem. 279, 22926â22933 (2004)
Stephan, M. M., Chen, M. A., Penado, K. M. & Rudnick, G. An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism. Biochemistry 36, 1322â1328 (1997)
Smicun, Y., Campbell, S. D., Chen, M. A., Gu, H. & Rudnick, G. The role of external loop regions in serotonin transport. J. Biol. Chem. 274, 36058â36064 (1999)
Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811â818 (2004)
Guerrero, S. A., Hecht, H. J., Hofmann, B., Biebl, H. & Singh, M. Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems. Appl. Microbiol. Biotechnol. 56, 718â723 (2001)
Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849â861 (1999)
Collaborative Computational Project, No. 4, The CCP4 suite: program for protein crystallography. Acta Crystallogr. D 50, 760â763 (1994)
Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458â463 (1999)
Brünger, A. T. et al. Crystallography & NMR system: a new software suite for maclomolecular structure determination. Acta Crystallogr. D 54, 905â921 (1998)
Yernool, D., Boudker, O., Folta-Stogniew, E. & Gouaux, E. Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42, 12981â12988 (2003)
Acknowledgements
We appreciate the beamtime and the assistance of the personnel at beamline 8.2.2 of the Advanced Light Source and beamlines X4A, X12B and X29 of the National Synchrotron Light Source; B. Honig and L. Forrest for help with sequence alignment; M. A. Gawinowicz for mass spectrometry and free amino acid analysis; J. Moon for help with bacterial culture and crystallization; H. Furukawa for assistance with vector construction and light scattering experiments; O. Boudker for liposome reconstitution; and D. Yernool, O. Boudker and R. Ryan for comments on the manuscript. A.Y. is on leave from the Laboratory for Structural Biochemistry, RIKEN Harima Institute at SPring-8, Japan. S.K.S. is supported by an NIH NRSA postdoctoral fellowship. The work was supported by the NIH. E.G. is an investigator with the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The coordinates for the structure have been deposited in the Protein Data Bank under the accession code 2A65. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Supplementary information
Supplementary Notes
This file contains Supplementary Table S1 (data collection and phasing statistics), Supplementary Methods, Supplementary Figures S1-S5 and accompanying Supplementary Figure Legends. (PDF 2016 kb)
Rights and permissions
About this article
Cite this article
Yamashita, A., Singh, S., Kawate, T. et al. Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 437, 215â223 (2005). https://doi.org/10.1038/nature03978
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature03978
This article is cited by
-
Oligomeric organization of membrane proteins from native membranes at nanoscale spatial and single-molecule resolution
Nature Nanotechnology (2024)
-
Ion and lipid orchestration of secondary active transport
Nature (2024)
-
GABA transport cycle: beyond a GAT feeling
Nature Structural & Molecular Biology (2023)
-
The dopamine transporter antiports potassium to increase the uptake of dopamine
Nature Communications (2022)
-
Reconstitution of GABA, Glycine and Glutamate Transporters
Neurochemical Research (2022)