Mechanism of chloride interaction with neurotransmitter:sodium symporters

Abstract

Neurotransmitter:sodium symporters (NSS) have a critical role in regulating neurotransmission and are targets for psychostimulants, anti-depressants and other drugs. Whereas the non-homologous glutamate transporters mediate chloride conductance, in the eukaryotic NSS chloride is transported together with the neurotransmitter. In contrast, transport by the bacterial NSS family members LeuT, Tyt1 and TnaT is chloride independent. The crystal structure of LeuT reveals an occluded binding pocket containing leucine and two sodium ions, and is highly relevant for the neurotransmitter transporters. 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 (gamma-aminobutyric acid) transporter GAT-1 from rat brain (also called SLC6A1) 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.

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

“…Theoretical studies also have proposed a cation-binding site in the aspartate transporter Glt Ph , based on a Monte Carlo search of the structure with a water probe, the results of which were tested by electrophysiological measurements (52). In another case, the identification of a negatively charged side chain in a chloride-independent bacterial homolog led to the prediction of the chloride-binding site in mammalian chloride-dependent NSS transporters; this prediction subsequently was confirmed by biochemical measurements (53,54).…”

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

“…3, f and g). It is also noteworthy that the recent discovery (27)(28)(29) of residues involved in Cl Ϫ binding in GAT-1 and SERT suggests that residues Tyr-233 (TM2), Gln-473, and Ser-477 (TM6) together with Asn-509 and Ser-513 (TM7) are likely to perform equivalent roles in GlyT2 (Fig. 4a).…”

“…4b), which lacks the hydroxyl necessary for the coordination of the Cl Ϫ ion and may prevent Cl Ϫ binding. Because a hydroxyl at this relative position is highly conserved in SLC6 transporters (27)(28)(29), the S513I substitution is predicted to interfere with glycine transport by disrupting the both Cl Ϫ (directly) and Na ϩ (indirectly) interactions.…”

“…For modeling the GlyT2 chloride ion-binding site, Cl Ϫ was placed into our model at the coordinates of the carboxylate carbon of the LeuT Glu-290 side chain (27), based on the assumption that this position may be occupied by Cl Ϫ in homologous transporters (27)(28)(29). The residues involved in the coordination of Cl Ϫ ion are generally conserved within the SLC6 transporter family.…”

The Aromatic and Charge Pairs of the Thin Extracellular Gate of the γ-Aminobutyric Acid Transporter GAT-1 Are Differently Impacted by Mutation

“…The GABA concentration used for radioactive transport is generally far below the K m and was 23 nM (87.1 Ci/mMol), as compared with the K m value of 1-2 M for GAT-1-WT (26). The reason is that large amounts of radioactive substrate are required to obtain a signal in the presence of saturating concentrations of unlabeled substrate, in particular for mutants with an increased K m .…”