Abstract
The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the neurotransmitter:sodium symporter (NSS) family, Na(+) stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na(+) transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na(+), allowing intracellular substrate and Na(+) release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na(+) on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein. Here, we used cysteine accessibility to measure conformational changes of LeuT in Escherichia coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na(+), these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.