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Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

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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.

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Figure 1: Effect of Na + on LeuT dynamics.
Figure 2: Effect of alanine on LeuT dynamics.
Figure 3: The configuration of TM6–TM10 interactions induced by Na + binding cannot be matched by Li + binding.
Figure 4: Effect of S1 and S2 site mutations and of Li + on activity and dynamics.

Change history

  • 02 June 2011

    Fig. 2a was corrected.

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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.

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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.

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

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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

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