Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster

Ciara Frances Pugh, Brian Thomas DeVree, Solveig Gaarde Schmidt, Claus Juul Loland*

*Corresponding author for this work

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Abstract

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.

Original languageEnglish
Article number3811
JournalCells
Volume11
Issue number23
Number of pages15
ISSN2073-4409
DOIs
Publication statusPublished - 2022

Keywords

  • dopamine transporter
  • protein purification
  • molecular pharmacology
  • scintillation proximity assay
  • radioligand
  • dopamine binding
  • NEUROTRANSMITTER TRANSPORTERS
  • BINDING-SITES
  • COCAINE
  • PROTEINS
  • ANTIDEPRESSANTS
  • RECOGNITION
  • MECHANISMS
  • INHIBITOR
  • IBOGAINE
  • ANALOGS

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