西亚试剂：Substrate-modulated gating dynamics in a Na+-coupled neurot

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

Yongfang Zhao,1, 2, 4, 7 Daniel S. Terry,5, 7 Lei Shi,5, 6, 7 Matthias Quick,1, 2, 4 Harel Weinstein,5, 6 Scott C. Blanchard5 & Jonathan A. Javitch1, 2, 3, 4

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 gradient1, 2, 3, 4, 5, 6. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine1, as well as by antidepressants7. 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 protein8. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold9, 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 tryptophan23, and the Na+-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy24. 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 bundle22. 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 leucine15, 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 importance of a second substrate (S2) binding site within the extracellular vestibule15, 20. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30?? away, in a manner that allows the Na+ gradient to power the transport mechanism.