![]() Reduction of monoamine levels in brain has also been observed after chronic treatment with monoamine transporter inhibitors in mice ( Avni et al., 1975 Rattray, 1991 Wilson et al., 1996 Wu et al., 1997 Baumann et al., 1998 Volkow et al., 1999 Wang et al., 1999 Mateo et al., 2005). Indeed, depletion of DA stores in DAT knock-out mice is observed despite the fact that the DA synthesis rate was elevated by twofold. Deletion of the DAT gene in mice leads to significant neurochemical changes characterized by a dramatic reduction of intracellular DA stores (95% decrease of DA levels compared with wild-type mice) ( Giros et al., 1996 Jones et al., 1998). DAT is the molecular target for therapeutic agents used in the treatment of mental disorders such as ADHD and depression as well as for cocaine and amphetamine, which are highly addictive and are major substances of abuse worldwide ( Barker and Blakely, 1995 Amara and Sonders, 1998 Robbins and Everitt, 1999). Thus, reuptake through DAT is the most effective way to limit the lifetime of DA signaling in the brain ( Amara and Kuhar, 1993 Giros and Caron, 1993 Torres et al., 2003b Cragg and Rice, 2004). Consequently, dysfunctions in the DA system are believed to contribute to the development of several neurological and psychiatric conditions such as Parkinson's disease, dystonia, depression, schizophrenia, attention deficit/hyperactivity disorder (ADHD), Tourette's syndrome, and drug addiction ( Carlsson, 1987 Roth and Elsworth, 1995 Koob and Le Moal, 1997 Greengard, 2001 Adinoff, 2004).ĭA, released into the synaptic cleft, is transported back to the presynaptic terminal via the plasma membrane DA transporter (DAT). In the CNS, the neurotransmitter dopamine (DA) mediates a wide array of physiological functions including regulation of locomotor activity, cognitive processes, neuroendocrine secretion, and the control of motivated behaviors including emotion, affect, and reward mechanisms ( Carlsson, 1987 Greengard, 2001). 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. ![]() Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. 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. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein–protein interaction. ![]() Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. 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. ![]() Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |