Résumé
Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na+-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na+-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.
Background: The mechanism coupling substrate binding to transport in neurotransmitter: sodium symporters (NSSs) is poorly understood.
Results: Site-directed fluorescence quenching spectroscopy experiments on the NSS homologue LeuT reveal a structural intermediate preceding transition to the inward-open conformation.
Conclusion: Stability of the intermediate might represent a rate-limiting barrier in the transport mechanism.
Significance: The data add to our mechanistic understanding of Na+-coupled transport across lipid bilayers.