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Substrate-dependent proton antiport in neurotransmitter:sodium symporters

Nature Chemical Biology volume 6, pages 109–116 (2010)Cite this article

Abstract

Neurotransmitter-sodium symporters (NSS), targets for psychostimulants and therapeutic drugs, have a critical role in neurotransmission. Whereas eukaryotic NSS show chloride-dependent transport, bacterial NSS feature Cl−-independent substrate transport. Recently we showed that mutation of an acidic residue near one of the sodium ion–binding sites in LeuT of Aquifex aeolicus or Tyt1 of Fusobacterium nucleatum renders substrate binding and/or transport Cl− dependent. We reasoned that the negative charge—provided either by Cl− or by the transporter itself—is required for substrate translocation. Here we show that Tyt1 reconstituted in proteoliposomes is strictly dependent on the Na+ gradient and is stimulated by an inside negative membrane potential and by an inversely oriented proton gradient. Notably, Na+/substrate symport elicited H+ efflux, indicative of Na+/substrate symport–coupled H+ antiport. Mutations that render the transport phenotype Cl− dependent essentially abolish the pH dependence. We propose unifying features of charge balance by all NSS members with similar mechanistic features but different molecular solutions.

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Figure 1: Tyrosine uptake by intact E. coli cells.
Figure 2: Tyrosine uptake in proteoliposomes containing purified recombinant Tyt1.
Figure 3: Influence of membrane potential and pH gradient on Tyt1-mediated transport.
Figure 4: Substrate kinetics of Cl−-dependent and Cl−-independent Tyt1 variants.
Figure 5: Na+/substrate symport–coupled H+ antiport.
Figure 6: Schematic representation of the NSS transport cycle.
Figure 7: The proximities of Na+ and H+/Cl− binding sites in Na+/H+ antiport and Na+/Cl− symport.

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Acknowledgements

We thank B. Kanner for helpful discussion, M.A. Gawinovicz of the Columbia University Medical Center Protein Core Facility for the MALDI-TOF analysis of Tyt1 and L. Chung for technical assistance. Computations were performed on the Ranger at the Texas Advanced Computing Center (TG-MCB090022) and the computational infrastructure of the Institute for Computational Biomedicine at Weill Cornell Medical College. This work was supported by US National Institutes of Health grants DA022413 and DA17293 (J.A.J.), P01 DA012923 (H.W.), DA015170 (E.L.M.) and K99 DA023694 (L.S.).

Author information

Author notes

  1. Yongfang Zhao and Matthias Quick: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Yongfang Zhao, Matthias Quick & Jonathan A Javitch

  2. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Matthias Quick & Jonathan A Javitch

  3. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Jonathan A Javitch

  4. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA

    Matthias Quick & Jonathan A Javitch

  5. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York, USA

    Lei Shi, Ernest L Mehler & Harel Weinstein

  6. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York, USA

    Lei Shi & Harel Weinstein

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  1. Yongfang Zhao
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Contributions

Y.Z. and M.Q. designed and performed transport and binding studies; L.S. and E.L.M. designed, carried out, and analyzed the computational simulations and pKa calculations. H.W. and J.A.J. helped to design experiments and analyze data related to the computational and biochemical studies, respectively. Y.Z., M.Q., L.S., E.L.M., H.W. and J.A.J. contributed to writing and editing the manuscript.

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Correspondence to Jonathan A Javitch.

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Zhao, Y., Quick, M., Shi, L. et al. Substrate-dependent proton antiport in neurotransmitter:sodium symporters. Nat Chem Biol 6, 109–116 (2010). https://doi.org/10.1038/nchembio.284

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