Structural and mechanistic characterization of the human dopamine transporter (hDAT)
EMSL Project ID
51213
Abstract
Neurotransmitter sodium symporters (NSS) belong to the SLC6 family of solute carriers and play an essential role in neurotransmitter homeostasis throughout the body. The human dopamine transporter (hDAT), in particular, is involved in dopamine homeostasis and regulation of neurotransmission by clearing of dopamine from the extracellular space using secondary active transport. Dopamine is a monoamine chemical messenger essential for regulation of reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants, such as cocaine, target the primary substrate binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of reuptake leads to accumulation of dopamine in the synapse resulting in heightened signaling. hDAT is also implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the structural studies of hDAT are nonexistent. Instability of hDAT in detergent micelles has been a major limiting factor in its successful biochemical, biophysical, and structural characterization. We developed a robust strategy for expression and purification of functionally active hDAT employing baculovirus-mediated expression system. We are currently carrying out structural studies of hDAT using cryo-EM with an ultimate goal to deduce the mechanistic basis of modulation of dopamine transporter activity by allosteric ligands and high affinity inhibitors.
Project Details
Start Date
2019-12-20
End Date
2020-07-20
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members