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 -