Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster
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Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster. / Pugh, Ciara Frances; DeVree, Brian Thomas; Schmidt, Solveig Gaarde; Loland, Claus Juul.
In: Cells, Vol. 11, No. 23, 3811, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster
AU - Pugh, Ciara Frances
AU - DeVree, Brian Thomas
AU - Schmidt, Solveig Gaarde
AU - Loland, Claus Juul
PY - 2022
Y1 - 2022
N2 - The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [H-3]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (K-d) for [H-3]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.
AB - The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [H-3]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (K-d) for [H-3]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.
KW - dopamine transporter
KW - protein purification
KW - molecular pharmacology
KW - scintillation proximity assay
KW - radioligand
KW - dopamine binding
KW - NEUROTRANSMITTER TRANSPORTERS
KW - BINDING-SITES
KW - COCAINE
KW - PROTEINS
KW - ANTIDEPRESSANTS
KW - RECOGNITION
KW - MECHANISMS
KW - INHIBITOR
KW - IBOGAINE
KW - ANALOGS
U2 - 10.3390/cells11233811
DO - 10.3390/cells11233811
M3 - Journal article
C2 - 36497070
VL - 11
JO - Cells
JF - Cells
SN - 2073-4409
IS - 23
M1 - 3811
ER -
ID: 329867089