Chloride binding site of neurotransmitter sodium symporters

Abstract

Neurotransmitter:sodium symporters (NSSs) play a critical role in signaling by reuptake of neurotransmitters. Eukaryotic NSSs are chloride-dependent, whereas prokaryotic NSS homologs like LeuT are chloride-independent but contain an acidic residue (Glu290 in LeuT) at a site where eukaryotic NSSs have a serine. The LeuT-E290S mutant displays chloride-dependent activity. We show that, in LeuT-E290S cocrystallized with bromide or chloride, the anion is coordinated by side chain hydroxyls from Tyr47, Ser290, and Thr254 and the side chain amide of Gln250. The bound anion and the nearby sodium ion in the Na1 site organize a connection between their coordinating residues and the extracellular gate of LeuT through a continuous H-bond network. The specific insights from the structures, combined with results from substrate binding studies and molecular dynamics simulations, reveal an anion-dependent occlusion mechanism for NSS and shed light on the functional role of chloride binding.

Keywords: SLC6; X-ray crystallography; antidepressant; membrane transport; psychostimulant.

Fig. 1.

Leu binding kinetics of LeuT-E290S. (A) The S2 site in LeuT-E290S is intact. Saturation binding of ³H-Leu (149 Ci/mmol) was performed with 25 ng LeuT-E290S in the presence of 250 mM NaCl by means of the SPA. Data from two independent experiments were subjected to one-site binding global fitting with variable Hill slope, yielding a molar binding stoichiometry of 1.86 ± 0.09 Leu:LeuT-E290S with a K_d of 0.79 ± 0.05 µM. (B) Leu binding by LeuT-E290S is dependent on Cl⁻. Binding of ³H-Leu by 25 ng LeuT-WT (100 nM ³H-Leu) or LeuT-E290S (1 µM ³H-Leu) was measured in the presence of 250 mM NaCl (solid bars) or Na-gluconate (open bars) with the SPA. Data were normalized with respect to the activity measured in the presence of NaCl. Error bars represent SEM of triplicate determinations.

Fig. 2.

Ion dependence of LeuT-E290S activity. (A) Binding of 1 µM ³H-Leu in 800 mM Na-gluconate in the presence of increasing concentrations of choline-chloride (■) or KBr (▽) yielded EC₅₀ of 160 ± 29.8 and 192 ± 49.2 mM, respectively. (B) Anion dependence of the binding of ²²Na⁺ by LeuT-WT or LeuT-E290S. Binding of 2 µM ²²Na⁺ (19.1 Ci/L) was measured in the presence of increasing concentrations of choline-chloride or KBr. ²²Na⁺ binding by LeuT-WT did not reveal any dependence on the Cl⁻ (●) or Br⁻ (○) concentration, whereas fitting of Cl⁻-dependent ²²Na⁺ binding of LeuT-E290S yielded an EC₅₀ of 156.6 ± 29.1 mM for Cl⁻ (■) and an EC₅₀ for Br⁻ of 122.2 ± 20.3 mM (▽). (C) Binding of 2 µM ²²Na⁺ by LeuT-E290S was measured in 0–800 mM unlabeled Na-gluconate in the presence of saturating Cl⁻ (■) or Br⁻ (▽). Fitting the data to a single binding site equation revealed an EC₅₀ for Na⁺ of 58.4 ± 7.4 mM in the presence of Cl⁻ and 80.1 ± 10.4 mM in the presence of Br⁻. (D) Na⁺ and Cl⁻ dependence of Leu binding by LeuT-E290S; 1 µM ³H-Leu binding was measured in the presence of increasing concentrations of Na⁺ and Cl⁻ (■) or Na-gluconate in the presence of 800 mM choline-chloride (□). Error bars represent SEM of triplicate determinations. Kinetic constants were obtained by using nonlinear algorithms in SigmaPlot and are expressed as mean ± SD of the fit.

Fig. 3.

The substrate and ion binding sites in the two structures. (A) The substrate and ion binding sites in the LeuT-E290S-Br⁻ structure. Na⁺ ions are shown as gray spheres, and Br⁻ ions are shown in light pink. The bound Leu is shown as purple sticks, whereas the Br⁻ coordinating residues are depicted as slate sticks. A simulated annealing omit map at a 4.5σ contour level obtained from refined coordinates in the absence of the Br⁻ ion is shown as a pink mesh, confirming the position of the bound Br⁻ ion. An isomorphous difference map between LeuT-E290S-Br⁻ and 3GJC (18) is displayed at 4.0σ contour level (green mesh). The strongest positive peak is found at the position of Br⁻ in the Cl⁻ site in chain B. The adjacent peak is caused by a side chain shift in Phe31 (Fig. S3). (B) The substrate and ion binding sites in the LeuT-E290S-Cl⁻ structure. The Na⁺ ions, the substrate, and coordinating residues are depicted as in A. Cl⁻ is shown as a green sphere. The pink mesh depicts a simulated annealing omit map at a 3.0σ contour level produced from the refined coordinates in the absence of the Cl⁻ ion.

Fig. 4.

The impact of a negative charge on the interaction network near the Na1 and S1 binding sites revealed by MD simulations. A and B show the evolution of the distances between the closest heavy atoms of the residues at positions 250 and 290 in (A) the E290S mutant and (B) the WT in the course of the MD trajectories; the corresponding constructs are depicted in C–F. The curves in A (E290S mutant) are from simulations carried out in the presence or absence of Cl⁻ (lines in cyan and orange, respectively), and the curves in B are for the corresponding constructs of the deprotonated (cyan) or protonated (orange) Glu290 in WT. Note that, in A, the dotted curves are from simulations with the n-octyl-β-glucopyranoside bound in the S2 site and found to reflect a similar trend as seen in the absence of n-octyl-β-glucopyranoside (solid curves). (C) In the E290S mutant, a negative charge provided by the bound Cl⁻ ion is key in maintaining the interaction network near the Na1 and S1 binding sites. (D) In the WT, this function is fulfilled by the deprotonated Glu290. In the absence of such a negative charge, water molecules are seen to penetrate to this region in both the WT and the E290S mutant, interacting directly with the residue at position 290 as well as with Asn286 and Tyr47. This water entry results in an equivalent rotation of the Gln250 side chain in both (E) the E290S mutant and (F) the WT.

Fig. 5.

The network of interactions between the substrate and the extracellular gate. (A) Ligand binding sites and the extracellular gate in the new structure. The dashed green lines show hydrogen bonds, which are part of the substrate-Cl⁻-gate bridge, of which Cl⁻ (or the negatively charged Glu290) is an integral part. The gate is closed, and Arg30 does not interact with Gln250. Ligands are shown as spheres in Inset. (B) Ligand binding sites and the extracellular gate in the WT outward-occluded LeuT structure (Protein Data Bank ID code 2A65) (6). Here, the negative charge of Glu290 effectively replaces Cl⁻ and keeps the substrate-Cl⁻-gate bridge intact. The gate is open, and Arg30 hydrogen bonds to Gln250.