Abstract
The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.
Highlights
The presynaptic dopamine transporter (DAT) encoded by the SLC6A3 gene is responsible for Na+-dependent reuptake of extracellular dopamine released from the nerve terminals and is a key mediator of dopaminergic neurotransmission [1,2,3,4]
Poly-D-lysine hydrobromide and anti-HA-Biotin (High Affinity (3F10)) from rat IgG1 were purchased from Sigma-Aldrich. 35-mm uncoated No 1.5 coverslip-bottomed dishes were purchased from MatTek. pcDNA3-EGFP plasmid DNA was a gift from Doug Golenbock (Addgene plasmid # 13031). pcDNA3.1-GFP-DRD2 plasmid DNA was a gift from Dr Jean-Michel Arrang (Addgene plasmid # 24099). pcDNA3.1-GFP-DRD2 plasmid DNA was a gift from Dr Jean-Michel Arrang (Addgene plasmid # 24099). tagRFP-C1-RFP-HA-DAT, pEYFP-C1-yellow fluorescent protein (YFP)-HA-DAT R60A, and pEYFP-C1-YFP-HA-DAT W63A plasmid DNA were a gift from Dr Alexander Sorkin (Addgene plasmid # 90265, 90245, and 90246 respectively). pcDNA3.1-D2 dopamine receptor (D2DR)-3xHA was acquired from the cDNA Resource Center
Our targeting strategy [20,21,23] features an organic ligand IDT444 composed of (i) a high-affinity cocaine analog that enables specific binding to the cell surface DAT molecules, (ii) an 11-carbon alkyl spacer to enable access to the binding site, (iii) a PEG chain to impart the ligand with aqueous solubility, and (iv) the biotin end which is captured by commercially available streptavidin-conjugated quantum dot (Qdot) with the emission maximum at 605 nm (Qdot605Sav) (Fig 1a; refer to [20] for IDT444 synthesis details)
Summary
The presynaptic dopamine transporter (DAT) encoded by the SLC6A3 gene is responsible for Na+-dependent reuptake of extracellular dopamine released from the nerve terminals and is a key mediator of dopaminergic neurotransmission [1,2,3,4]. As there are no extracellular enzymes that catabolize dopamine upon its release at the presynaptic active zone, the duration of dopamine transient signal is primarily controlled by the rapid dopamine diffusion away from the active zone. Quantum dots reveal conformation-sensitive diffusion heterogeneity of dopamine transporter to the extrasynaptic areas and subsequent reuptake of dopamine through the perisynaptic DATs [1,2,10]. Considering the slow transport cycle of DAT (1–5 dopamine molecules/s) [10] relative to the rapid timescale of synaptic dopamine signals, proper DAT membrane localization is a key prerequisite for efficient dopamine clearance
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