Abstract
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.
| Original language | English (US) |
|---|---|
| Article number | e0200085 |
| Journal | PLoS One |
| Volume | 13 |
| Issue number | 7 |
| DOIs | |
| State | Published - Jul 1 2018 |
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ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)
Cite this
Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation. / Navratna, Vikas; Tosh, Dilip K.; Jacobson, Kenneth A.; Gouaux, Eric.
In: PLoS One, Vol. 13, No. 7, e0200085, 01.07.2018.Research output: Contribution to journal › Article
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TY - JOUR
T1 - Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation
AU - Navratna, Vikas
AU - Tosh, Dilip K.
AU - Jacobson, Kenneth A.
AU - Gouaux, Eric
PY - 2018/7/1
Y1 - 2018/7/1
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0200085
DO - 10.1371/journal.pone.0200085
M3 - Article
C2 - 29965988
AN - SCOPUS:85049374822
VL - 13
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 7
M1 - e0200085
ER -