Technical Reference #3289

3289.

Modulation of the dopamine transporter by interaction with Secretory Carrier Membrane Protein 2 Anja W. Fjorback; Heidi K. Müller; Jana Haase; Merete K. Raarup; Ove Wiborg, University Hospital Aarhus, Biochemical and Biophysical Research Communications, 406(3289), (2011)
Link To Paper

Abstract:
The monoamine transporters for dopamine (DAT); norepinephrine (NET) and serotonin (SERT) facilitate the homeostatic balance of neurotransmitters in the synaptic cleft and thus; play a fundamental role in regulating neuronal activity. Despite the importance of these monoamine transporters in controlling brain function; only relatively little information is available regarding the cellular and molecular regulation of these proteins. The monoamine transporters have been found to associate with a number of different proteins that regulate the function and subcellular localization of the transporters. We recently reported a functional interaction between SERT and the Secretory Carrier Membrane Protein 2 (SCAMP2). Here; we demonstrate that SCAMP2 also plays a role in the functional regulation of DAT. DAT and SCAMP2 interaction is here verified by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) microscopy. Moreover; co-expression of DAT and SCAMP2 results in a decrease in DAT-mediated dopamine uptake caused by reduced levels of DAT molecules on the cell surface. Our finding that SCAMP2 interacts with and regulates the subcellular distribution of both DAT and SERT suggests that interaction with SCAMP2 may constitute an important mechanism for coordinating cell surface expression of monoamine transporters.

Keywords:
<div><font face=AdvGulliv-R size=1 color=black>Dopamine; Serotonine; Protein interaction; FRET; SCAMP2</font></div>

Materials & Methods:
Confocal microscopy HEK-293 and SHSY5Y cells were grown on glass coverslips (#1.5 MENZEL-GLASER; GmbH) or glass-bottom dishes (MatTek Corporation P35G-1.5–20-C) and transfected at 50% confluence with CFP-SCAMP2/YFP-DAT/ – or CFP-only/YFP-only FRET pairs. Live HEK-293 and SHSY5Y cells were imaged on a Zeiss confocal LSM 510 META microscope at 37 C using a 40 NA 1.2 C-Apochromat objective. Sensitized acceptor emission FRET measurements and analysis were performed according to the PFRET procedure developed by the group of Wallrabe et al. [19] on CFP-SCAMP2/YFP-DAT (HEK-293 and SHSY5Y) or CFP-only/YFP-only samples. Donor and acceptor fluorescence signals were detected following excitation with 144 lW of 458 nm (donor) and 14 lW of 514 nm (acceptor) laser power as measured at the sample with a 10 Plan-Neofluar objective. For removal of bleed through and cross talk; images for samples transfected with donor or acceptor only were acquired. Images were 256 256 (CFP-only/YFP-only) or 512 512 (CFPSCAMP2/ YFP-DAT) pixels in size and were acquired with pixel dwell time 3.2 ls; line average 2; 0.11 lm/pixel; 8-bit intensity resolution. The CFP and YFP fluorescence signals were detected in the 468–500 nm and 535–575 nm ranges; using the META detector with gains set to 600 (amplifier gain one). For quantification of the sensitized acceptor emission FRET images the ImageJ-based PFRET plugin of Wallrabe et al. was applied [19]. For our calculations it was assumed that G = 1. FRET efficiency values (Eapp) were calculated on a pixel-by-pixel basis (for Eapp images) and for automatically defined regions of interests (ROI’s) of size 5 5 pixels. Only ROI’s having signal levels P25 for 80% of pixels within the ROI were included for Eapp analysis (lower bounds). Lower bounds for signal levels used in bleedthrough and direct acceptor excitation corrections were set to 25. FRET experiments were performed two (CFP-only/YFP-only) or three (CFP-SCAMP2/YFP-DAT) times with independent transfections; each time acquiring 13–20 images with 1–2 cells/image. For CFP-only/YFP-only the image size was reduced to 256 256 pixels; only partially covering a cell; in order to avoid contributions from close-lying neighbouring cells with variable expression levels; and due to the much higher number of Eapp values obtained from the homogeneous CFP-only/YFP-only cellular distribution.

Microscopic Technique
Confocal microscopy

Cell Type(s)
HEK-293 and SHSY5Y cells