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The Mechanism of a Neurotransmitter:Sodium Symporter—Inward Release of Na+ and Substrate Is Triggered by Substrate in a Second Binding Site
Lei Shi,1,2,7 Matthias Quick,3,6,7 Yongfang Zhao,3 Harel Weinstein,1,2 and Jonathan A. Javitch3,4,5,6,
1 Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
2 HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
3 Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA
4 Department of Psychiatry, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA
5 Department of Pharmacology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA
6 Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY 10032, USA
Summary
Eukaryotic neurotransmitter:sodium symporters (NSSs), targets for antidepressants and psychostimulants, terminate neurotransmission by sodium-driven reuptake. The crystal structure of LeuTAa, a prokaryotic NSS homolog, revealed an occluded state in which one leucine and two Na+ ions are bound, but provided limited clues to the molecular mechanism of transport. Using steered molecular dynamics simulations, we explored the substrate translocation pathway of LeuT. We identified a second substrate binding site located in the extracellular vestibule comprised of residues shown recently to participate in binding tricyclic antidepressants. Binding and flux experiments showed that the two binding sites can be occupied simultaneously. The substrate in the secondary site allosterically triggers intracellular release of Na+ and substrate from the primary site, thereby functioning as a “symport effector.” Because tricyclic antidepressants bind differently to this secondary site, they do not promote substrate release from the primary site and thus act as symport uncouplers and inhibit transport.
