The mechanism of a neurotransmitter:sodium symporter--inward release of Na+ and substrate is triggered by substrate in a second binding site.
Eukaryotic neurotransmitter:sodium symporters (NSSs), targets for antidepressants and psychostimulants, terminate neurotransmission by sodium-driven reuptake. The crystal structure of LeuT(Aa), 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.
Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Lei
L
Shi
Matthias
M
Quick
Yongfang
Y
Zhao
Harel
H
Weinstein
Jonathan A
JA
Javitch
D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D002450:Cell Communication; D003198:Computer Simulation; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism
D050480:Plasma Membrane Neurotransmitter Transport Proteins; D011994:Recombinant Proteins; D012965:Sodium Chloride; D012964:Sodium; D007930:Leucine