Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes

Abstract

The dopamine transporter (DAT) is one of the key regulators of dopamine (DA) signaling in the central nervous system (CNS) and in the periphery. Recent reports in a model of Parkinson's disease (PD) have shown that dopamine neuronal loss in the CNS impacts the expression of DAT in peripheral immune cells. The mechanism underlying this connection is still unclear but could be illuminated with sensitive and high-throughput detection of DAT-expressing immune cells in the circulation. Herein, we have developed fluorescently labeled ligands (FLL) that bind to surface-expressing DAT with high affinity and selectivity. The diSulfoCy5-FLL (GC04-38) was utilized to label DAT in human and mouse peripheral blood mononuclear cells (PBMCs) that were analyzed via flow cytometry. Selective labeling was validated using DAT KO mouse PBMCs. Our studies provide an efficient and highly sensitive method using this novel DAT-selective FLL to advance our fundamental understanding of DAT expression and activity in PBMCs in health and disease and as a potential peripheral biomarker.

The Structural Basis of the Activity Cliff in Modafinil-Based Dopamine Transporter Inhibitors

“…Bradford protein assay (Bio-Rad, Hercules, CA, USA) was used to determine the protein concentration and the membranes were diluted to 1000 μg/mL and stored in a −80 °C freezer for later use. Radioligand binding assays were conducted similar to those previously described [ 44 , 45 ]. Experiments were conducted in 96-well polypropylene plates containing 50 μL of various concentrations of the test compound, diluted using 30% DMSO vehicle, 300 μL of binding buffer (50 mM Tris, 120 mM NaCl, pH 7.4), 50 μL of [ 3 H]WIN35,428 [ 46 ] (final concentration 1.5 nM; K d = 38.1 nM; NOVANDI Chemistry AB, 78 Ci/mmol SA), and 100 μL of membranes 30 μg/well).…”