Visualization of the Cocaine-Sensitive Dopamine Transporter with Ligand-Conjugated Quantum Dots

Abstract

1À3 Impaired DA signaling has been linked to a number of neurodegenerative and psychiatric disorders such as attention-deficit hyperactivity disorder (ADHD), bipolar disorder, major depression, Tourette's syndrome, Parkinson's disease, and schizophrenia. 4À8 The synaptic DA concentration influences postsynaptic DA signal transduction capacity and is modulated by the activity of a presynaptic D 2 DA receptor, that modulates DA release, and the DA transporter (DAT), 1,9 that clears DA to achieve DA inactivation and recycling.10 DAT (SLC6A3) is a member of a family of Na þ -coupled solute transporters whose substrates include neurotransmitters, nutrients, osmolytes, and amino acids. Several reports have demonstrated that experimental DAT deficiency results in pronounced changes in dopaminergic tone and locomotor hyperactivity.10À12 In addition, DAT is the primary target for widely used psychostimulants, such as amphetamine and cocaine that acutely elevate synaptic DA concentrations. Cocaine is a competitive DAT inhibitor and attenuates DA clearance by occupying the DA binding site on DAT, whereas amphetamine promotes DAT-mediated DA efflux that also results in the increased DA synaptic concentration. 13 DAT activity has also been demonstrated to be a subject of acute, dynamic regulation by several post-translational mechanisms, such as constitutive endocytosis, protein-kinase-C (PKC)-dependent internalization, proteinÀprotein interactions, and substrate-induced changes in surface expression level. 13,14 The spatial organization and temporal control of these mechanisms remain largely unknown and, when disrupted, may influence risk for disorders linked to compromised DA signaling.The investigation of DAT regulation has thus far trailed similar efforts directed at membrane receptors and channels due a number of important challenges. First, the lack of an efficient antibody against an extracellular epitope does not allow direct localization and visualization of DAT molecules in living cells without prior chemical processing (fixation and permeabilization). 15,16 Second, the use of popular fusion tags, such as green fluorescent protein (GFP) and hemagglutinin (HA), requires genetic perturbation of DAT and thus does not allow direct visualization of endogenous DAT. Third, traditional autoradiographic, biochemical, and optical techniques to monitor DAT expression, function, and cellular distribution suffer from suboptimal 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...

Single-Quantum-Dot Tracking Reveals Altered Membrane Dynamics of an Attention-Deficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant

“…38,40 The flexible PEG-based linker of IDT444 also reduces potential steric hindrance of QDs with the cell surface in addition to enhancing water solubility of the ligand. 46 With this probe strategy, we demonstrated previously that QD-IDT444 conjugates successfully detected plasma membrane-localized DATs in both transiently and stably transfected expression systems.…”

“…The location of the R615C substitution in a highly conserved region of the transporter’s C-terminus is shown. (Reproduced with permission from ref 38. Copyright 2011 American Chemical Society.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…To visualize the localization of MA transporters in membrane microdomains of intact cells, investigators have turned to fluorescent colocalization studies, targeting in particular the location of transporters in membrane microdomains that are enriched Kinase Modulation of Biogenic Amine Transport for GM1 ganglioside, which can be labeled using fluorescently tagged cholera toxin B subunit (Navaroli et al, 2011;Sakrikar et al, 2012). Transporters are most often visualized using antibodies-based detection strategies, although in some cases transporters have been labeled with fluorescently tagged small molecule transporter ligands (Chang et al, 2012;Eriksen et al, 2009;Kovtun et al, 2011).…”

Functional properties of dopaminergic neurons obtained from fibroblasts of a patient with PARK2 form of Parkinson’s disease

“…С помощью вестерн-блоттинга был исследован уро-вень белка DAT в культурах дофаминергических нейро-нов, полученных от пациента -носителя мутаций в гене PARK2 и от здорового донора; в качестве контроля были проанализированы лизаты клеток линии HeLa, поскольку в клетках данной опухолевой линии присутствует DAT [17]. Как видно из рис.…”