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

Autor: Guthrie DA; Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States., Klein Herenbrink C; Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark., Lycas MD; Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark., Ku T; Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States., Bonifazi A; Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States., DeVree BT; Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark., Mathiasen S; Department of Psychiatry, Columbia University Vagelos College of Physicians & Surgeon and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, United States., Javitch JA; Department of Psychiatry, Columbia University Vagelos College of Physicians & Surgeon and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, United States., Grimm JB; Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, United States., Lavis L; Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, United States., Gether U; Department of Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark., Newman AH; Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States.
Jazyk: angličtina
Zdroj: ACS chemical neuroscience [ACS Chem Neurosci] 2020 Oct 21; Vol. 11 (20), pp. 3288-3300. Date of Electronic Publication: 2020 Oct 01.
DOI: 10.1021/acschemneuro.0c00397
Abstrakt: 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 (JF 549 and JF 646 ). High affinity binding of the resulting ligands to DAT was demonstrated by potent inhibition of [ 3 H]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 JF 549 -conjugated DG3-80 ( 3 ) and JF 646 -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 ( d STORM) 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.
Databáze: MEDLINE
Externí odkaz: