Monitoring conformational changes in the human neurotransmitter transporter homologue LeuT with ¹⁹F-NMR spectroscopy

Neurotransmitter:sodium symporters (NSS) reuptake neurotransmitter molecules from the synaptic space through Na⁺-coupled transport. They are thought to work via the alternating access mechanism, exploring multiple configurations dictated by the binding of substrates and ions. Much of the current knowledge about these transporters has been derived from examining the structure of the Leucine Transporter (LeuT), a bacterial counterpart to human NSSs. Multiple crystal structures of LeuT provided valuable information regarding the steps involved in this mechanism. Dynamical data connecting the crystal structure to the transport cycle are critical to understanding how ligands are translated through the membrane. In the present study, we applied ¹⁹F-based nuclear magnetic resonance (NMR) spectroscopy to ¹⁹F labelled LeuT to monitor how substrates and ions binding affect the conformations of the transporter. By selecting mutations and ligands known to affect the conformational equilibrium of LeuT, we identified and assigned four NMR resonances to specific conformational states of LeuT. We observe that Na⁺ ions produce closure of the extracellular vestibule to a state similarly induced by Na⁺ and substrates. Conversely, K⁺ ions seem to shift the conformational equilibrium toward inward-facing intermediates, arguably by competing with Na⁺. The present study assembles a framework for NMR-based dynamical studies of NSS transporters and demonstrates its feasibility for tackling large membrane LeuT-fold transporters.