Chloride binding site of neurotransmitter sodium symporters

Abstract

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog

“…The connection between this salt bridge and Na1 involved Gln-250 in TM6 and Glu-290 in TM7 (Fig. 9, B and C), as described previously (51). In simulations of WT outward-occluded LeuT, for example, Gln-250 formed negligible direct interactions with Arg-30 ( Fig.…”

The Two Na+ Sites in the Human Serotonin Transporter Play Distinct Roles in the Ion Coupling and Electrogenicity of Transport

“…Evidence for two Na ϩ -binding sites in monoamine transporters was recently bolstered by the solving of the eukaryotic Na ϩ /Cl Ϫ -dependent dopamine transporter from Drosophila melanogaster (dDAT) with sodium-binding sites comparable with those in LeuT (12). Crystal structures of dDAT and a LeuT Cl Ϫ -dependent mutant (E290S) (30) have greatly advanced our understanding of the Cl Ϫ -binding site in these proteins and support previous biochemical studies (31)(32)(33).…”

Arginine oscillation explains Na + independence in the substrate/product antiporter CaiT

“…Although the eukaryotic neurotransmitter:sodium symporters including GAT-1 are chloride-dependent, their bacterial counterparts LeuT, Tyt1, and TnaT are chloride-independent. Introduction of a glutamate near the putative Na1 binding site in GAT-1 renders this transporter chloride-independent, whereas the reciprocal mutations make LeuT and Tyt1 chloride-dependent (29)(30)(31). Similarly, replacement of an aspartate by threonine transformed the lightdriven proton pump bacteriorhodopsin into a chloride pump (32).…”