Overview of the structure and function of the dopamine transporter and its protein interactions

Abstract

The dopamine transporter (DAT) plays an integral role in dopamine neurotransmission through the clearance of dopamine from the extracellular space. Dysregulation of DAT is central to the pathophysiology of numerous neuropsychiatric disorders and as such is an attractive therapeutic target. DAT belongs to the solute carrier family 6 (SLC6) class of Na⁺/Cl^- dependent transporters that move various cargo into neurons against their concentration gradient. This review focuses on DAT (SCL6A3 protein) while extending the narrative to the closely related transporters for serotonin and norepinephrine where needed for comparison or functional relevance. Cloning and site-directed mutagenesis experiments provided early structural knowledge of DAT but our contemporary understanding was achieved through a combination of crystallization of the related bacterial transporter LeuT, homology modeling, and subsequently the crystallization of drosophila DAT. These seminal findings enabled a better understanding of the conformational states involved in the transport of substrate, subsequently aiding state-specific drug design. Post-translational modifications to DAT such as phosphorylation, palmitoylation, ubiquitination also influence the plasma membrane localization and kinetics. Substrates and drugs can interact with multiple sites within DAT including the primary S1 and S2 sites involved in dopamine binding and novel allosteric sites. Major research has centered around the question what determines the substrate and inhibitor selectivity of DAT in comparison to serotonin and norepinephrine transporters. DAT has been implicated in many neurological disorders and may play a role in the pathology of HIV and Parkinson's disease via direct physical interaction with HIV-1 Tat and α-synuclein proteins respectively.

Keywords: allosteric modulators; dopamine transporter; inward-open; oligomerization; outward-open; post translational modifications; tat; α-synuclein.

FIGURE 1

Topological representation of DAT. The TM helices, intracellular loops and extracellular loops are labeled. The orange spheres represent the Na⁺ ions. Green and white spherical model represents the substrate molecule. Two inverted triangles represent the two domains with an inverted axis of symmetry that contributes to substrate binding pocket.

FIGURE 2

Different conformational states of hDAT during the transport cycle. The TM helices are numbered and represented by tubes. Important residues involved in conformational changes are shown in stick model and labeled. Substrate molecule (DA) is represented in spherical model. (A). In the outward-open state (apo form), the salt bridge between D476 (TM10) and R85 (TM1) which acts as the first extracellular gate is interrupted. Similarly, the interaction between Y156 and F320 which function as the second extracellular gate is also broken. At the intracellular side, there are strong ionic interactions between E448-K257, R445-E428 and D436-R60 residues. Particularly, D436-R60 acts as the intracellular gate keeper. (B). In the outward-open substrate bound state, a DA molecule binds at the orthostatic binding site. All the ionic interactions are the same as in the apo form. (C). In the substrate-occluded state, the extracellular gates, D476-R85 and Y156 and F320 are closed. There is also movement of the EL4 loop to close the extracellular vestibule. (D). In the inward-open state, the ionic interactions E448-K257, R445-E428 are interrupted, and the intracellular gate formed by D436-R60 is opened. (E). In the inward-open state (apo from), DA is released into the intracellular sites and the cycle is reset.

FIGURE 3

Post-Translational Modification (PTM) sites for hDAT are depicted. TM helices are labeled, and color coded. The sites of PTM are shown in spherical models and are color coded. Magenta spheres represent sites for palmitoylation, red spheres represent ubiquitination sites, cyan spheres represent sites for glycosylation, and green spheres represent the site for PKC-mediated endocytosis.

FIGURE 4

Binding poses for the DA, cocaine and KM822 molecules at the orthosteric (S1), S2, and allosteric site respectively. The ligand molecules are shown by the stick model with a transparent surface. The 2D ligand interactions map is also shown for the ligands.