Differential regulation of the serotonin transporter by vesicle-associated membrane protein 2 in cells of neuronal versus non-neuronal origin

TY - JOUR

T1 - Differential regulation of the serotonin transporter by vesicle-associated membrane protein 2 in cells of neuronal versus non-neuronal origin

AU - Müller, Heidi Kaastrup

AU - Kragballe, Marie

AU - Fjorback, Anja Winther

AU - Wiborg, Ove

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pone.0097540

DO - 10.1371/journal.pone.0097540

M3 - Journal article

C2 - 24878716

SN - 1932-6203

VL - 9

SP - e97540

JO - PLOS ONE

JF - PLOS ONE

IS - 5

ER -