Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

An open gate to neurotransmitters Neurotransmitters are removed from the synapse by neurotransmitter/Na + symporters (NSSs) in a reuptake process driven by the Na + gradient. Zhao et al . examined the coupling of substrate binding to structural transitions in the prokaryotic NSS homologue LeuT. Using single-molecule fluorescence resonance energy transfer, functional experiments and computational studies, they find that LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. Neurotransmitter/Na + symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na + electrochemical gradient 1 , 2 , 3 , 4 , 5 , 6 . NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine 1 , as well as by antidepressants 7 . The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na + are bound deep within the protein 8 . This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 . Subsequently, an ‘outward-open’ conformation was determined in the presence of the inhibitor tryptophan 23 , and the Na + -dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy 24 . In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle 22 . We investigated how substrate binding is coupled to structural transitions in LeuT during Na + -coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ∼10-fold faster than leucine 15 , 25 , we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional impo