Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site

Crystal structure of LeuT Neurotransmitter sodium-coupled symporters (NSSs) couple the uptake of a neurotransmitter with one or more sodium ions, removing the neurotransmitter from the synaptic cleft. LeuT, a prokaryotic orthologue of the NSS family, has been used to explore the relationship between molecular mechanism and atomic structure in a broad range of transporters. There is some controversy over whether there are one or two high-affinity substrate-binding sites in LeuT. The initial crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity substrate-binding site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the second (S2) site is thought to be located within the extracellular vestibule. In this paper, the authors perform direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. The authors conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site.