TY - JOUR
T1 - Novel Fluorescent Ligands Enable Single-Molecule Localization Microscopy of the Dopamine Transporter
AU - Guthrie, Daryl A.
AU - Klein Herenbrink, Carmen
AU - Lycas, Matthew Domenic
AU - Ku, Therese
AU - Bonifazi, Alessandro
AU - Devree, Brian T.
AU - Mathiasen, Signe
AU - Javitch, Jonathan A.
AU - Grimm, Jonathan B.
AU - Lavis, Luke
AU - Gether, Ulrik
AU - Newman, Amy Hauck
N1 - Publisher Copyright:
© 2020 American Chemical Society. All rights reserved.
PY - 2020/10/21
Y1 - 2020/10/21
N2 - 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.
AB - 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.
KW - Dopamine
KW - dSTORM
KW - Fluorescent Ligands
KW - Microscopy
KW - Single Particle Tracking
KW - Transporters
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U2 - 10.1021/acschemneuro.0c00397
DO - 10.1021/acschemneuro.0c00397
M3 - Article
C2 - 32926777
AN - SCOPUS:85094219654
SN - 1948-7193
VL - 11
SP - 3288
EP - 3300
JO - ACS chemical neuroscience
JF - ACS chemical neuroscience
IS - 20
ER -