Identification of a chloride ion binding site in Na ⁺ /Cl ⁻ -dependent transporters

Abstract

The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl ؊ independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl ؊ ions. However, the only Cl ؊ ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK As and homology modeling to predict the location of a functionally important Cl ؊ binding site in serotonin transporter and other Cl ؊ -dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl ؊ ion and through the observation of sequence conservation patterns in other Cl ؊ -dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl ؊ and Na ؉ ions during transport. Other implications of the model are also discussed.neurotransmitters ͉ neurotransmitter:sodium symporters ͉ serotonin ͉ homology modeling ͉ pK A calculation

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).…”

Binding of the Amphetamine-like 1-Phenyl-piperazine to Monoamine Transporters

“…16 The model includes the two sodium ions close to the binding site but does not include a chloride ion, which was proposed to be placed in the vicinity of the binding site of chloride-dependent transporters. 30,31 The presence of this ion was shown to neither influence the predicted binding of the native ligand of hSERT in docking calculations nor affect the placement of PP in test computations. The protein was initially prepared using the Protein Preparation facility in the Schrodinger suite of software and has been previously described for hSERT.…”

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).…”

“…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.…”