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Single-molecule dynamics of gating in a neurotransmitter transporter homologue

Abstract

Neurotransmitter:Na+ symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na+ gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechanism that couples ion and substrate binding to transport.

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Figure 1: Structural landmarks and the disposition of the engineered Cys pairs in the crystal structure of LeuT.
Figure 2: Single-molecule imaging of LeuT.
Figure 3: The structural context of the observed dynamic changes.
Figure 4: Effects of mutation and CMI on FRET histograms of LeuT-H7C/R86C and LeuT-239C/480C.
Figure 5: Long single-molecule trajectories reveal FRET transitions.

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Acknowledgements

We thank R. Altman for assistance with reagents for single-molecule experiments; F. Carvalho for the preparation of membranes; J. Munro for help measuring anisotropy; R. Dave for preliminary photobleaching optimization studies; and M. Quick for helpful discussion and comments on the manuscript. Molecular graphic figures and movies were prepared with PyMOL (DeLano Scientific; http://www.pymol.org). 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.), and DA023694 (L.S.). D.S.T. is supported by the Tri-Institutional Training Program in Computational Biology and Medicine.

Author information

Authors and Affiliations

Authors

Contributions

Y.Z. expressed, purified, labelled and functionally characterized the LeuT mutants. Y.Z. and D.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

Correspondence to Scott C. Blanchard or Jonathan A. Javitch.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Figures 1-8 with legends, Supplementary Tables 1-3, Notes for Supplementary Movies 1-2 and a Reference. (PDF 1324 kb)

Supplementary Movie 1

This flash movie shows a side view of conformational changes in the simulated transport mechanism from a ~180 ns molecular dynamics simulation that induces and equilibrates the inward-open structure (orange) starting from the LeuT crystal structure (gray). The conformational changes agree with and are reflected in the results from the smFRET measurements. (SWF 9327 kb)

Supplementary Movie 2

This flash movie shows an intracellular view of conformational changes in the simulated transport mechanism from a ~180 ns molecular dynamics simulation that induces and equilibrates the inward-open structure (orange) starting from the LeuT crystal structure (gray). The conformational changes agree with and are reflected in the results from the smFRET measurements. (SWF 9399 kb)

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Zhao, Y., Terry, D., Shi, L. et al. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465, 188–193 (2010). https://doi.org/10.1038/nature09057

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