title: Visualization of the cocaine-sensitive dopamine transporter with ligand-conjugated quantum dots.
abstract: The presynaptic dopamine (DA) transporter is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinson's disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporter's regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of efficient DAT-specific fluorescent probes. We developed a quantum dot-based labeling approach that uses a DAT-specific, biotinylated ligand, 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444), that can be bound by streptavidin-conjugated quantum dots. Flow cytometry and confocal microscopy were used to detect DAT in stably and transiently transfected mammalian cells. IDT444 is useful for quantum-dot-based fluorescent assays to monitor DAT expression, function, and plasma membrane trafficking in living cells as evidenced by the visualization of acute, protein-kinase-C (PKC)-dependent DAT internalization.
authors: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, USA.
authors: Oleg
authors: O
authors: Kovtun
authors: Ian D
authors: ID
authors: Tomlinson
authors: Dhananjay S
authors: DS
authors: Sakrikar
authors: Jerry C
authors: JC
authors: Chang
authors: Randy D
authors: RD
authors: Blakely
authors: Sandra J
authors: SJ
authors: Rosenthal
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002477:Cells / Q000378:metabolism / Q000648:ultrastructure; D003042:Cocaine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000187:drug effects* / Q000648:ultrastructure*; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008024:Ligands; D018613:Microscopy, Confocal; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D045663:Quantum Dots; D019809:Streptavidin / Q000737:chemistry
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D007202:Indicators and Reagents; D008024:Ligands; D010703:Phorbol Esters; D019809:Streptavidin; D011493:Protein Kinase C; D003042:Cocaine