Abstract
The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake.
Highlights
The serotonin transporter (SERT) is well-known for its involvement in brain serotonin (5-HT) homeostasis as it determines the magnitude and duration of serotonergic signalling [1,2]
Lysates from HEK293 MSR cells transfected with SERT were incubated with fusion proteins of GST and the full-length vesicle-associated membrane protein 2 (VAMP2), synaptosome-associated protein of 25 kDa (SNAP25) and syntaxin 1A
The results revealed a direct interaction between VAMP2 and SERT and confirmed the previously reported interaction between syntaxin 1A and SERT
Summary
The serotonin transporter (SERT) is well-known for its involvement in brain serotonin (5-HT) homeostasis as it determines the magnitude and duration of serotonergic signalling [1,2]. The control of SERT transport capacity via intrinsic and trafficking mediated events is subject to regulation by various mechanisms including modulation by transporter substrates and antagonists, signaling molecules and to a large extent by direct protein-protein interactions [5]. This is emphasized by the diverse nature of SERT interacting proteins identified to date, which includes proteins involved in neurotransmitter release, cellular signaling, trafficking through the secretory pathway and presynaptic targeting. Syntaxin 1A binds to the N-terminal of SERT and regulates both transporter trafficking and 5-HT transport activity through mechanisms that involve subcellular redistribution and changes in the transport stoichiometry of SERT [6,9,10]
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