AbstractThe human dopamine transporter(hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the molecular architecture of hDAT and its various conformational states are poorly understood. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, first we identified ligands that stabilize hDAT in detergent micelles. Then, we screened ∼200 single residue mutants of hDAT using high-throughput scintillation proximity assay, and identified a thermostable variant(I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.
Chemogenetic activation of mesoaccumbal Gamma-Aminobutyric Acid projections selectively tunes responses to predictive cues when reward value is abruptly decreased