Substrate-Induced Conformational Change in LeuT

Abstract

LeuT is a prokaryotic amino acid transporter that has been used extensively as a model for neurotransmitter transport. We recently demonstrated that the conformational change induced by Na+ ions requires the Na2 site observed in LeuT crystal structures. We observed this conformational change using LeuT in E. coli membranes, in the absence of detergent, by a decrease in reactivity of a single cysteine (Y265C) in the cytoplasmic permeation pathway. We now show that this effect of Na+ is observed whether K+ or NMDG+ is used as a control ion. In the presence of Na+, addition of alanine, a substrate, induces the reverse conformational change, opening the cytoplasmic pathway and increasing Cys-265 reactivity. Three mutations in the substrate binding site each altered the affinity of LeuT for leucine and alanine, but did not interfere with the conformational change induced by Na+. One of the mutations also blocked the substrate-dependent conformational change. The results suggest a mechanism by which substrate interactions with the central binding site of LeuT reverse the ability of Na+ to stabilize outward-facing conformations of this model transporter.