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Mechanism of chloride interaction with neurotransmitter:sodium symporters

Nature volume 449, pages 726–730 (2007)Cite this article

Abstract

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs1,2. Whereas the non-homologous glutamate transporters mediate chloride conductance3, in the eukaryotic NSS chloride is transported together with the neurotransmitter4,5,6,7. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent8,9,10. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions9, and is highly relevant for the neurotransmitter transporters11,12,13. However, the precise role of chloride in neurotransmitter transport and the location of its binding site remain elusive. Here we show that introduction of a negatively charged amino acid at or near one of the two putative sodium-binding sites of the GABA (γ-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1)14,15 renders both net flux and exchange of GABA largely chloride independent. In contrast to wild-type GAT-1, a marked stimulation of the rate of net flux, but not of exchange, was observed when the internal pH was lowered. Equivalent mutations introduced in the mouse GABA transporter GAT4 (SLC6A11) and the human dopamine transporter DAT (SLC6A3) also result in chloride-independent transport, whereas the reciprocal mutations in LeuT and Tyt1 render substrate binding and/or uptake by these bacterial NSS chloride dependent. Our data indicate that the negative charge, provided either by chloride or by the transporter itself, is required during binding and translocation of the neurotransmitter, probably to counterbalance the charge of the co-transported sodium ions.

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Figure 1: A putative chloride site in GAT-1.
Figure 2: Net flux by wild-type and chloride-independent mutant transporters: theory and experiment.
Figure 3: Transport and binding by other NSS transporters.
Figure 4: Anion selectivity of GAT-1 S331 mutants and putative chloride-binding site in the NSS.

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Acknowledgements

This work was supported by a grant from the Israel Science Foundation (to B.I.K.) and NIH grants (to J.A.J.). We thank S. Qu for the generation of the GAT-1 S331G mutant; M. Sonders for making the LeuT E290S and Tyt1 A263S mutants; and L. Chung for cell culture and membrane preparations.

Author Contributions E.Z., A.B. and B.I.K. did the experimental work for the eukaryotic transporters and M.Q. and Y.Z. did the experiments on the bacterial transporters. E.Z., M.Q., J.A.J. and B.I.K. did project planning and manuscript writing.

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Authors and Affiliations

  1. Department of Biochemistry, Hebrew University Hadassah Medical School, POB 12272, Jerusalem 91120, Israel,

    Elia Zomot, Annie Bendahan & Baruch I. Kanner

  2. Center for Molecular Recognition and,,

    Matthias Quick, Yongfang Zhao & Jonathan A. Javitch

  3. Departments of Psychiatry and Pharmacology, Columbia University, 630 W. 168th, New York, New York 10032, USA,

    Jonathan A. Javitch

  4. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, USA,

    Matthias Quick & Jonathan A. Javitch

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  1. Elia Zomot
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Correspondence to Baruch I. Kanner.

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Zomot, E., Bendahan, A., Quick, M. et al. Mechanism of chloride interaction with neurotransmitter:sodium symporters. Nature 449, 726–730 (2007). https://doi.org/10.1038/nature06133

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