Abstract
Neurotransmitter/Na+ symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na+ electrochemical gradient1,2,3,4,5,6. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine1, as well as by antidepressants7. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na+ are bound deep within the protein8. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold9,10,11,12,13,14,15,16,17,18,19,20,21,22. Subsequently, an ‘outward-open’ conformation was determined in the presence of the inhibitor tryptophan23, and the Na+-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy24. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle22. We investigated how substrate binding is coupled to structural transitions in LeuT during Na+-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine15,25, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule15,20. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30 Å away, in a manner that allows the Na+ gradient to power the transport mechanism.
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
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
02 June 2011
Fig. 2a was corrected.
References
Amara, S. G. & Sonders, M. S. Neurotransmitter transporters as molecular targets for addictive drugs. Drug Alcohol Depend. 51, 87–96 (1998)
Rudnick, G. Mechanisms of Biogenic Amine Neurotransmitter Transporters 2nd edn (Humana, 2002)
Sonders, M. S., Quick, M. & Javitch, J. A. How did the neurotransmitter cross the bilayer? A closer view. Curr. Opin. Neurobiol. 15, 296–304 (2005)
Gu, H., Wall, S. C. & Rudnick, G. Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. J. Biol. Chem. 269, 7124–7130 (1994)
Torres, G. E., Gainetdinov, R. R. & Caron, M. G. Plasma membrane monoamine transporters: structure, regulation and function. Nature Rev. Neurosci. 4, 13–25 (2003)
Krause, S. & Schwarz, W. Identification and selective inhibition of the channel mode of the neuronal GABA transporter 1. Mol. Pharmacol. 68, 1728–1735 (2005)
Iversen, L. Neurotransmitter transporters and their impact on the development of psychopharmacology. Br. J. Pharmacol. 147 (Suppl 1). S82–S88 (2006)
Yamashita, A. et al. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437, 215–223 (2005)
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. Mol. Pharmacol. 70, 1630–1642 (2006)
Quick, M. et al. 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 (2006)
Forrest, L. R. et al. Mechanism for alternating access in neurotransmitter transporters. Proc. Natl Acad. Sci. USA 105, 10338–10343 (2008)
Kniazeff, J. et al. An intracellular interaction network regulates conformational transitions in the dopamine transporter. J. Biol. Chem. 283, 17691–17701 (2008)
Noskov, S. Y. Molecular mechanism of substrate specificity in the bacterial neutral amino acid transporter LeuT. Proteins 73, 851–861 (2008)
Noskov, S. Y. & Roux, B. Control of ion selectivity in LeuT: two Na+ binding sites with two different mechanisms. J. Mol. Biol. 377, 804–818 (2008)
Shi, L. et al. The mechanism of a neurotransmitter:sodium symporter — inward release of Na+ and substrate is triggered by substrate in a second binding site. Mol. Cell 30, 667–677 (2008)
Singh, S. K. LeuT: a prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure. Channels (Austin) 2, 380–389 (2008)
Crisman, T. J., Qu, S., Kanner, B. I. & Forrest, L. R. Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats. Proc. Natl Acad. Sci. USA 106, 20752–20757 (2009)
Khalili-Araghi, F. et al. Molecular dynamics simulations of membrane channels and transporters. Curr. Opin. Struct. Biol. 19, 128–137 (2009)
Li, J. & Tajkhorshid, E. Ion-releasing state of a secondary membrane transporter. Biophys. J. 97, L29–L31 (2009)
Quick, M. et al. Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation. Proc. Natl Acad. Sci. USA 106, 5563–5568 (2009)
Shi, L. & Weinstein, H. Conformational rearrangements to the intracellular open states of the LeuT and ApcT transporters are modulated by common mechanisms. Biophys. J. 99, L103–L105 (2010)
Zhao, Y. et al. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465, 188–193 (2010)
Singh, S. K., Piscitelli, C. L., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008)
Claxton, D. P. et al. Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters. Nature Struct. Mol. Biol. 17, 822–829 (2010)
Singh, S. K., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007)
Beckett, D., Kovaleva, E. & Schatz, P. J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999)
Zhou, Z. et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317, 1390–1393 (2007)
Zhou, Z. et al. Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures. Nature Struct. Mol. Biol. 16, 652–657 (2009)
Sen, N., Shi, L., Beuming, T., Weinstein, H. & Javitch, J. A. A pincer-like configuration of TM2 in the human dopamine transporter is responsible for indirect effects on cocaine binding. Neuropharmacology 49, 780–790 (2005)
Piscitelli, C. L., Krishnamurthy, H. & Gouaux, E. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site. Nature 468, 1129–1132 (2010)
Schaffner, W. & Weissmann, C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal. Biochem. 56, 502–514 (1973)
Munro, J. B., Altman, R. B., O'Connor, N. & Blanchard, S. C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505–517 (2007)
Caplan, D. A., Subbotina, J. O. & Noskov, S. Y. Molecular mechanism of ion-ion and ion-substrate coupling in the Na+-dependent leucine transporter LeuT. Biophys. J. 95, 4613–4621 (2008)
Acknowledgements
We thank R. Altman for assistance in preparing reagents for single-molecule experiments and F. Carvalho for the preparation of membranes. Molecular graphics were prepared with PyMOL. Computations were performed on Ranger at the Texas Advanced Computing Center (TG-MCB090022) and the David A. Cofrin computational infrastructure of the Institute for Computational Biomedicine at Weill Cornell Medical College. This work was supported in part by National Institutes of Health grants DA17293 and DA022413 (J.A.J.), DA12408 (H.W.), DA023694 (L.S.), the Irma T. Hirschl/Monique Weill-Caulier trusts (S.C.B.) and the Lieber Center for Schizophrenia Research and Treatment. D.S.T. is supported by the Tri-Institutional Training Program in Computational Biology and Medicine.
Author information
Authors and Affiliations
Contributions
Y.Z. expressed, purified and labelled the LeuT mutants. M.Q. and Y.Z. performed the functional characterization of the mutants. Y.Z. and D.S.T. designed, carried out and analysed the single-molecule experiments; L.S. and H.W. designed and analysed the computational studies, which were carried out by L.S.; S.C.B. and J.A.J. helped to design the biochemical and single-molecule experiments and, with L.S. and H.W., helped to interpret the data. All the authors contributed to writing and editing the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
The file contains a Supplementary Discussion, Supplementary Figures 1-10 with legends, Supplementary Table 1 and additional references. (PDF 1171 kb)
Rights and permissions
About this article
Cite this article
Zhao, Y., Terry, D., Shi, L. et al. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474, 109–113 (2011). https://doi.org/10.1038/nature09971
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature09971
This article is cited by
-
Multi-parameter photon-by-photon hidden Markov modeling
Nature Communications (2022)
-
Serotonin Transporter Ala276 Mouse: Novel Model to Assess the Neurochemical and Behavioral Impact of Thr276 Phosphorylation In Vivo
Neurochemical Research (2022)
-
Insight into the direct interaction of Na+ with NhaA and mechanistic implications
Scientific Reports (2021)
-
X-ray structure of LeuT in an inward-facing occluded conformation reveals mechanism of substrate release
Nature Communications (2020)
-
The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner
Neurochemical Research (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.