Abstract
Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.
Highlights
Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport
We have sought to quantify the allosteric coupling between the conformational changes observed by single-molecule fluorescence resonance energy transfer (smFRET) and the binding of various ligands in terms of thermodynamic coupling, which manifests as either changes in the occupancy of states or the rate of transitions between those states[23]
In previous smFRET investigations of LeuT, we have shown that movement of its intracellular N-terminal segment away from the 12 transmembrane segments (TMs) bundle is associated with substrate-modulated intracellular gating[15,16]
Summary
Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Most eukaryotic NSS are comprised of 12 transmembrane segments (TMs), whereas prokaryotic homologs can have 12 or 11 TMs, with two inverted structurally related repeats (TMs 1–5 and TMs 6–10)[1,2] These secondary active transporters couple the movement of substrates and Na+ across the bilayer, using the physiological Na+ gradient to concentrate substrates within the cell[3]. We and others have reported that LeuT retains comparable binding activity[17,18,19] and exhibits similar conformational states[20] in detergent and liposomes With this smFRET approach, we are able to perform time-dependent measurements of energy transfer efficiency between donor and acceptor fluorophores that are site- attached to LeuT, and thereby monitor the changes in intramolecular distance between them, with sub-nanometer accuracy[21,22]. We have sought to quantify the allosteric coupling between the conformational changes observed by smFRET and the binding of various ligands in terms of thermodynamic coupling, which manifests as either changes in the occupancy of states or the rate of transitions between those states[23]
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