Molecular Mechanism of Ion-Ion and Ion-Substrate Coupling in the Na+-Dependent Leucine Transporter LeuT

Abstract

Ion-coupled transport of neurotransmitter molecules by neurotransmitter:sodium symporters (NSS) play an important role in the regulation of neuronal signaling. One of the major events in the transport cycle is ion-substrate coupling and formation of the high-affinity occluded state with bound ions and substrate. Molecular mechanisms of ion-substrate coupling and the corresponding ion-substrate stoichiometry in NSS transporters has yet to be understood. The recent determination of a high-resolution structure for a bacterial homolog of Na(+)/Cl(-)-dependent neurotransmitter transporters, LeuT, offers a unique opportunity to analyze the functional roles of the multi-ion binding sites within the binding pocket. The binding pocket of LeuT contains two metal binding sites. The first ion in site NA1 is directly coupled to the bound substrate (Leu) with the second ion in the neighboring site (NA2) only approximately 7 A away. Extensive, fully atomistic, molecular dynamics, and free energy simulations of LeuT in an explicit lipid bilayer are performed to evaluate substrate-binding affinity as a function of the ion load (single versus double occupancy) and occupancy by specific monovalent cations. It was shown that double ion occupancy of the binding pocket is required to ensure substrate coupling to Na(+) and not to Li(+) or K(+) cations. Furthermore, it was found that presence of the ion in site NA2 is required for structural stability of the binding pocket as well as amplified selectivity for Na(+) in the case of double ion occupancy.

Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model

“…This result is entirely expected, because two ions residing in sites Na2 and Na3 are separated by only about 8 Ǻ, and unfavorable electrostatic repulsion between the two ions will affect ionbinding affinity at these sites. This combination of low-affinity and high-affinity ion-binding sites is similar to that of the leucine transporter LeuT, suggesting different functionality for the highaffinity and low-affinity sites (19).…”

“…A similar scheme had been used previously for studies of substrate and antidepressant binding to the neurotransmitter transporter LeuT (23). The absolute binding free energies for ions were evaluated from FEP experiments with periodic boundary conditions using parameters developed earlier (19). The weighted histogram analysis method was used to postprocess the FEP data and evaluate ion-binding affinities for the binding sites (Na1, Na2, and Na3).…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…592 itors (Singh et al, 2007Zhou et al, 2009). It is noteworthy that these LeuT structures have been established as valid templates for molecular modeling of the mammalian NTTs, and LeuT is emerging as an important model protein for biophysical and computational studies addressing the molecular dynamics underlying NTT function and pharmacology (Jørgensen et al, 2007a(Jørgensen et al, , 2008Caplan et al, 2008;Celik et al, 2008a;Noskov and Roux, 2008;Noskov, 2008;Shi et al, 2008;Shaikh and Tajkhorshid, 2010;Zhao et al, 2010a,b).…”

“…MD simulations in combination with biochemical and biophysical experiments have been used to study conformational changes taking place during substrate and ion binding, and the subsequent translocation processes in both LeuT andSLC6 (Forrest et al, 2007, 2008;Jørgensen et al, 2007a;Beuming et al, 2008;Caplan et al, 2008;Celik et al, 2008a;Kniazeff et al, 2008;Noskov and Roux, 2008;Noskov, 2008;Shi et al, 2008;Tavoulari et al, 2009;Zhao et al, 2010a,b). However, the time scale of typical MD simulations (10 -100 ns) is far from the turnover rates of membrane transporters (0.1-10 s), and MD simulations are unlikely to be able to simulate the structural rearrangements during a complete translocation cycle.…”

Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site

“…These structures reveal that outwardly facing transporters possess an aqueously accessible external vestibule that opens to a compact central/primary substrate-binding site (S1) composed of residues from TM domains 1, 3, 6, and 8 (15). Binding of substrate, two Na ϩ (Na1 and Na2) ions, and one Cl Ϫ ion to their respective sites triggers conformational changes leading to closure of an extracellular gate that occludes S1, followed by opening of the intracellular gate and generation of an inwardly facing form that releases solutes to the cytoplasm (14,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The S1 site is highly conserved in mammalian transporters, and mutagenesis of many DAT residues near this site, including Phe-76, Asp-79, Val-152, Phe-155, Tyr-156, Asn-157, Phe-319, Val-327, and Ser-421, reduces dopamine transport (31)(32)(33)(34)(35)(36)(37)(38), supporting the participation of these amino acids in substrate recognition or translocation.…”