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

The neurotransmitter dopamine (DA) modulates a variety of physiological functions and behavioral responses including attention, arousal, cognition, reward, and motor activity in the central nervous system (Figure ​(Figure11).1−3 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.4−8 The synaptic DA concentration influences postsynaptic DA signal transduction capacity and is modulated by the activity of a presynaptic D2 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+-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.10−12 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, protein–protein 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. Figure 1 Structures of DAT and its relevant substrates. A two-dimensional topology of DAT based on the leucine transporter (LeuT) is shown with 12 transmembrane segments, intracellularly oriented N- and C-termini, and substrate binding site. Structures of dopamine, ... 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 spatial and temporal resolution and are limited to providing ensemble-averaged information.17,18 Recently, a series of dye-conjugated fluorescent cocaine analogues has been developed and successfully used to directly visualize DAT in living cells for the first time. Cha, Eriksen and colleagues used an organic dye-conjugated 2β-carbomethoxy-3β-(3,4-dichlorophenyl)tropane (RTI 111) ligand to visualize changes in DAT cellular movement in response to different stimuli via laser confocal microscopy.16 However, this ligand does not have the photostability properties to permit single-molecule resolution. We have focused on developing new DAT-specific ligands for conjugation with nanometer-sized semiconductor nanocrystals, known as quantum dots (Qdots). Qdots offer several distinct advantages over conventional fluorophores and permit visualization of membrane-associated proteins with high accuracy and temporal resolution, with reported values as low as 10 nm with 10 ms integration time.19−23 Specifically, their excellent brightness and superior resistance to photodegradation enable noninvasive imaging of complex biological processes with high signal-to-noise ratio (SNR) over time scales from milliseconds to hours. Also, the broad absorption spectra and size-dependent, narrow, symmetric emission spectra of Qdots considerably simplify multiplexed, molecular imaging experiments. We have previously reported the synthesis of GBR12909- and GBR12935-based DAT-specific ligands for conjugation with Qdots.24,25 In this effort, we sought to improve the design of the DAT ligand by incorporating a phenyltropane-based dopamine reuptake inhibitor parent compound (β-CFT, WIN 35,428) into the structure. β-CFT is a structural analogue of cocaine, is 3–10× more potent than cocaine, and is characterized by excellent structural stability.26,27 Our choice of the parent compound is also validated by multiple instances of the use of radiolabeled β-CFT to map DAT distribution in the animal and human brain.28−31 Here, we present a relatively simple and rapid approach for Qdot-based direct visualization of DAT in living cells that uses a DAT-specific, biotinylated 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444) in conjunction with streptavidin-conjugated Qdots (SavQdots). Using this approach, we demonstrate the specificity of DAT Qdot labeling and the ability to detect DAT-expressing mammalian cells at a combination of low nanomolar concentrations of IDT444 and picomolar concentrations of Qdots. To determine whether we could use our Qdot-based approach to capture DAT trafficking, we visualized acute, PKC-dependent internalization of DAT-Qdot complexes in response to phorbol ester treatment. Finally, we show the advantages of Qdot photophysical properties in time-lapse image series acquisition over extended periods of time.