The vesicular monoamine transporter (VMAT) is responsible for the storage of all monoamine neurotransmitters in the nervous system and inhibition of VMAT by the drug reserpine causes a behavioral state resembling depression. Intracellular trafficking has been proposed to regulate VMAT by controlling its localization to two types of secretory vesicles: synaptic vesicles (SVs) that cluster at the active zone of nerve terminals, and large dense core vesicles (LDCVs) that perform a neuromodulatory role. Although trafficking motifs for VMAT have been identified in culture, the signals required for its localization to SVs and LDCVs in vivo remain unclear. Furthermore, it is not known how trafficking signals in either VMAT or any other neurotransmitter transporter effects either localization or function in an intact animal. We propose to use the model genetic organism Drosophila melanogaster to explore these questions. We have characterized a mutation in the endogenous dVMAT gene, which provides a useful background for the analysis of transgenes containing trafficking mutants. We also have identified two splice variants (DVMAT-A and B) that contain divergent C-terminal trafficking domains and a motif in the C-terminus of DVMAT-A required for endocytosis in vitro. We will now determine how this motif and other potential endocytosis signals contribute to the localization of DVMAT-A to SVs in vivo. We also will identify signals required to sort DVMAT-A to LDCVs. To determine whether the C-terminal trafficking domain of DVMAT-A is required for its function in vivo, we will compare the ability of DVMAT-A, DVMAT-B and a C-terminal truncation to rescue defects in dVMAT mutants that depend on the function of DVMAT-A. Mutation of dVMAT also prevents histamine storage in subretinal glia that express DVMAT-B, thus providing an assay to study the function of this unusual isoform in vivo. We will perform genetic rescue experiments to determine whether the C-terminus of DVMAT-B is required for its function in vivo. To further investigate how the novel trafficking domain in DVMAT-B contributes to its function in vivo, we will determine its subcellular localization in the subretinal glia. These experiments will help determine how a neurotransmitter transporter linked to neuropsychiatric illness is regulated in vivo. The results may aid the development of novel treatment strategies for depression.
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