X-ray structure of dopamine transporter elucidates antidepressant mechanism

Abstract

Antidepressants targeting Na(+)/Cl(-)-coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses.

Figure 1. Architecture of dDAT_cryst

a, Structure of dDAT_cryst viewed parallel to membrane. Nortriptyline, sodium ions, a chloride ion, and a cholesterol molecule are shown in sphere representation in magenta, purple, green, and yellow, respectively. b, View of dDAT_cryst from the extracellular face. c, Surface representation showing that ligand and ion binding sites are accessible from the extracellular vestibule. Nortriptyline and TMs 1, 3, 6, and 8 are colored as in panel a.

Figure 2. Antidepressant binding site

a, Overall view of the nortriptyline-bound dDAT_crystFo-Fc densities (blue mesh) for drug and ions are contoured at σ levels of 2.5 and 3.0, respectively. b, Close-up view of the drug binding pocket. Na⁺ and Cl ions are shown as spheres. Nortriptyline is represented as sticks (magenta). The amino group of nortriptyline is 2.8 Å from the carbonyl oxygen of F43 (TM1a) and the N-methyl group of nortriptyline is 3.2 Å from the carbonyl oxygen of F319. Residues lining the drug binding pocket with interfacial areas greater than 10 Ų are represented as sticks. c. Comparison of the drug or substrate-binding pocket of dDAT_cryst with that of LeuT (PDB id. 2A65), respectively. The distance between the carboxylate group of leucine and Tyr 108 (spheres) is 2.7 Å in the occluded state (2A65) and 5.1 Å in the inhibitor-bound state (3F3A) of LeuT, whereas the equivalent interaction in dDAT_cryst between Asp 46 and Tyr 124 is 3.2 Å.

Figure 3. Ion binding sites

a, Na1 and chloride ion-binding sites. Na⁺ is purple and Cl is green and both are modeled as spheres. b, Coordination at the Na2 site. Coordination at the site is trigonal bipyramidal with the water molecule (red sphere) 3.3 Å from the sodium ion. Distances are in ängstroms for residues that are in the coordination sphere and interactions are shown by dashed lines. Residues are colored according to their respective TMs.

Figure 4. Cholesterol site

a, Cholesterol (yellow sticks) shown with Fo-Fc density (light magenta) contoured at 2.0 σ. Residues that interface with the cholesterol group are represented as sticks. b, Potential role of cholesterol in maintaining an outward-open state of transporter. Cholesterol (sticks with transparent surface) sterically clashes with the position of TM1a in the inward-open conformation of LeuT (PDB id. 3TT3).

Figure 5. Extracellular and cytoplasmic gates and the C-terminal latch

a, Relative locations of the open extracellular gate (red box), closed cytoplasmic gate (blue box), and C-terminal latch (green box) in dDAT_cryst. b, The width of the extracellular gate is depicted by the distances between Y124 and F319 (10 Å), and R52 and D475 (10 Å). Nortriptyline, ions, and helices are colored as in figure 1. c, The cytoplasmic gate is closed by polar and electrostatic interactions between TM1a, TM2, IL1, TM6b, and TM8. d, The C-terminal helix following TM12 is bound to the cytoplasmic face of the transporter via polar interactions with IL1. Polar and electrostatic bonds are represented as gray dashed lines.

Figure 6. Mechanisms of antidepressants and cholesterol

a, The TCA nortriptyline (magenta) wedges between scaffold helices 3 and 8 and the core helices 1 and 6, preventing the movement of TMs 1b and 6a from closing the extracellular vestibule. b, Cholesterol (yellow) is bound in an intracellular pocket and prevents the movement of TM1a from opening, thereby stabilizing the outward-open conformation of DAT. The C-terminal latch interacts with IL1 as part of the cytoplasmic gate.