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Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter.

ABSTRACT: The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and is the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF₅₄₉ and JF₆₄₆). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [³H]dopamine uptake in DAT transfected CAD cells and by competition radioligand binding experiments on rat striatal membranes. Visualization of binding was substantiated by confocal or TIRF microscopy revealing selective binding of the analogues to DAT transfected CAD cells. Single particle tracking experiments were performed with JF₅₄₉-conjugated DG3-80 (3) and JF₆₄₆-conjugated DG4-91 (4) on DAT transfected CAD cells enabling quantification and categorization of the dynamic behavior of DAT into four distinct motion classes (immobile, confined, Brownian, and directed). Finally, we show that the ligands can be used in direct stochastic optical reconstruction microscopy (dSTORM) experiments permitting further analyses of DAT distribution on the nanoscale. In summary, these novel fluorescent ligands are promising new tools for studying DAT localization and regulation with single-molecule resolution.

SUBMITTER: Guthrie DA

PROVIDER: S-EPMC8283659 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter.

Guthrie Daryl A DA Klein Herenbrink Carmen C Lycas Matthew Domenic MD Ku Therese T Bonifazi Alessandro A DeVree Brian T BT Mathiasen Signe S Javitch Jonathan A JA Grimm Jonathan B JB Lavis Luke L Gether Ulrik U Newman Amy Hauck AH

ACS chemical neuroscience 20201001 20

The dopamine transporter (DAT) is critical for spatiotemporal control of dopaminergic neurotransmission and is the target for therapeutic agents, including ADHD medications, and abused substances, such as cocaine. Here, we develop new fluorescently labeled ligands that bind DAT with high affinity and enable single-molecule detection of the transporter. The cocaine analogue MFZ2-12 (1) was conjugated to novel rhodamine-based Janelia Fluorophores (JF549 and JF646). Hig ...[more]

PMID: 32926777

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Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter.

Publications

Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter.

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