Physical and Functional Interaction between the Dopamine Transporter and the Synaptic Vesicle Protein Synaptogyrin-3

Abstract

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pulldown assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the nonneuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the proteinprotein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Amphetamine Action at the Cocaine- and Antidepressant-Sensitive Serotonin Transporter Is Modulated by αCaMKII

“…The generation of ␣CaMKII knock-out (KO) mice has been described previously (Elgersma et al, 2002). SERT-Cre mice were used to produce SERT KO mice and are described in detail in Zhuang et al (2005) and Montgomery et al (2014).…”

“…FRET was measured with a Carl Zeiss Axiovert 200 epifluorescence microscope. We used either HEK-293 cells or rat hippocampal neurons transiently transfected with plasmid cDNA (1.7 g) by means of the calcium phosphate coprecipitation method, as described previously (Egaña et al, 2009;Sucic et al, 2010). Cells were transfected directly in Ibidi -slide chambered coverslips with 8 wells.…”

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…Disrupting this interaction in vivo leads to hyperlocomotion in mice, suggesting impaired DA uptake and/or D 2 receptor signaling . The DAT may be indirectly influenced by VMAT2 through interaction with the synaptic vesicle protein synaptogyrin-3 (Egaña et al, 2009). Increasing synaptogyrin-3 expression in vitro increases DAT function, which is blocked by treatment with the VMAT2 inhibitor reserpine (Egaña et al, 2009).…”

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

“…The homoassociation of more than one transporter molecule of the NSS family has been shown by biochemical approaches such as co-immunoprecipitation of differently epitope-tagged SERT and DAT constructs (5), oxidative crosslinking of SERT (6) and DAT (7,8) and also by optical measurements like Förster resonance energy transfer (FRET) for DAT (9,10), GAT1 and SERT (11)(12)(13), and GLYT (14).…”