Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site

Abstract

Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

Figure 1. Leu binding measured by ITC and equilibriumdialysis

a, b, ITC data for Leu binding to wild-type LeuT (a) and Leu binding to mutant Tyr 108 Phe–LeuT^K (see Methods) (b). Raw injection heats (expressed as differential power) are shown in the top panels and the corresponding specific binding isotherms (calculated from the integrated injection heats and normalized to moles of injectant) are shown in the bottom panels, determined at 25 °C and pH 7.0. Square brackets denote concentration. c, d, Quantitation of [³H]Leu-binding stoichiometry by equilibrium dialysis for the wild type (open circle, solid line) or the Leu 400 Ala mutant (open triangle, dashed line) (c), and for Tyr 108 Phe-LeuT^K (d). Errors bars, s.e.m.; n = 2.

Figure 2. Leu binding measured by scintillation proximity assays

a, Saturation binding isotherms and nonlinear regression analysis for wild-type LeuT (open circle, solid line), Leu 400 Ala mutant (open triangle, dashed line) and Leu 400 Cys mutant (open square, dotted line). c.p.m., counts per minute. b, Saturation binding at high LeuT concentration (~20K_d), quantifying substrate-binding stoichiometry. Symbols and lines are as in a. c, Saturation binding for wild-type LeuT in the absence (same data as in a) or presence of 1mM clomipramine (closed circle, dashed line). Error bars, s.e.m.; n = 2.

Figure 3. Transport kinetics of [³H]Ala uptake

a, Steady-state Ala uptake as a function of Ala concentration at pH 5. Inset, the corresponding Eadie–Hofstee plot with linear regression (r² = 0.93). Error bars, s.e.m.; n = 4. b, Steady-state Ala uptake at pH 5 in the presence of valinomycin to induce a membrane potential. Inset, the corresponding Eadie–Hofstee plot with linear regression (r² = 0.96). Error bars, s.e.m.; n = 2. S, substrate.

Figure 4. LeuT mechanism

Starting fromthe apo transporter in an open-tooutside conformation (a), substrate (S) and sodium ions bind, forming the outward-facing occluded conformation (b) characterized by closure of a `thin gate' over the S1 substrate-binding site. Clomipramine, which inhibits transport, binds in the extracellular vestibule, directly above the thin gate, near the putative S2 site. The substrate- and ion-bound transporter undergoes structural isomerization to form the inward-facing conformation (c), allowing release of substrate and ions to the intracellular solution, thereby generating an open-to-inside apo transporter (d) that isomerizes to the open-to-outside conformation (a).