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

PY - 2020

Y1 - 2020

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

U2 - 10.1021/acschemneuro.0c00397

DO - 10.1021/acschemneuro.0c00397

M3 - Journal article

C2 - 32926777

AN - SCOPUS:85094219654

VL - 11

SP - 3288

EP - 3300

JO - ACS Chemical Neuroscience

JF - ACS Chemical Neuroscience

SN - 1948-7193

IS - 20

ER -