Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation

• Published in: PloS one Vol. 13; no. 7; p. e0200085
• Main Authors: Navratna, Vikas, Tosh, Dilip K, Jacobson, Kenneth A, Gouaux, Eric
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02.07.2018; Public Library of Science (PLoS)
• Subjects: Active transport; Allosteric properties; Allosteric Regulation; Attention deficit hyperactivity disorder; Autism; Binding sites; Biology and Life Sciences; Cell Membrane - metabolism; Cocaine; Conformation; Crystallization; Diabetes; Dopamine; Dopamine - metabolism; Dopamine Plasma Membrane Transport Proteins - antagonists & inhibitors; Dopamine Plasma Membrane Transport Proteins - chemistry; Dopamine Plasma Membrane Transport Proteins - isolation & purification; Dopamine Plasma Membrane Transport Proteins - metabolism; Dopamine transporter; Dopaminergic mechanisms; Emotional factors; Gene expression; Genetic aspects; Health aspects; HEK293 Cells; High-Throughput Screening Assays; Homeostasis; Humans; Inhibition (psychology); Inhibitors; Kidney diseases; Laboratories; Ligands; Micelles; Motor task performance; Mutagenesis; Mutagenesis, Site-Directed; Mutants; Mutation; Neurodegeneration; Neurological diseases; Neurotransmission; Organic chemistry; Physical Sciences; Protein Conformation; Protein Stability; Proteins; Purification; Reinforcement; Research and Analysis Methods; Serotonin; Structural analysis; Temperature; Transport proteins; Trends; Oregon; United States > US; Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0200085

The 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 structural studies of hDAT have proven difficult. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, we identified ligands that stabilize hDAT in detergent micelles. We then screened ~200 single residue mutants of hDAT using a 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.