1. Department of Biochemistry, Hebrew University Hadassah Medical School, POB 12272, Jerusalem 91120, Israel
2. Center for Molecular Recognition and,
3. Departments of Psychiatry and Pharmacology, Columbia University, 630 W. 168th, New York, New York 10032, USA
4. Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, USA

Correspondence to: Baruch I. Kanner¹ Correspondence and requests for materials should be addressed to B.I.K. (Email: kannerb@cc.huji.ac.il).

Abstract

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs^{1, 2}. Whereas the non-homologous glutamate transporters mediate chloride conductance³, in the eukaryotic NSS chloride is transported together with the neurotransmitter^{4, 5, 6, 7}. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent^{8, 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 transporters^{11, 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 (-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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