The sequestration of neurotransmitters, such as serotonin, norepinephrine, dopamine, and histamine into intracellular vesicles for subsequent release is a fundamental cellular event in neurons and secreting cells. Reduced or aberrant activity of the monoamine translocator of the synaptic vesicle may play a central role in Parkinson's Disease. Reserpine, used clinically for many years to control hypertension, causes the depletion of amines from the storage vesicles by inhibition of the monoamine translocator in the vesicle membrane. The regulation of uptake of monoamine neurotransmitters into storage vesicles may play an important role in affective psychological disorders related to responses has a paracrine action in the gut, and is a neurotransmitter in the central nervous system. The strategy of this proposal is based on the rationale that identification of the reserpine binding site on monoamine transporters will provide a basic understanding of the biochemical mechanism of action of monoamine sequestration into vesicles and the factors which regulate the activity of the translocator. The binding site for reserpine and reserpine-like compounds on monoamine vesicle translocators will be mapped. This work will be accomplished in four Specific Aims: (1) a series of novel radioiodinated and tritiated reserpine and tetrabenazine photoaffinity probes will be developed; (2) photolabels will be functionally assessed as inhibitors of monoamine uptake in chromaffin granules and in reconstituted vesicles containing pure recombinant baculovirus-Sf9-expressed translocators; (3) overexpression and large- scale purification of the synaptic vesicle transporter (SVAT), the chromaffin granule transporter (CGAT), and the mast cell histamine transporter (HIST) will be performed; purification strategies will be based on polyhistidine epitope tagging, lectin affinity chromatography, and ion exchange chromatography; (4) photolabeled peptides will be prepared and identified by combined strategies of chemical and tic cleavage, purification by HPLC, SDS-PAGE, and/or monoclonal and peptide- specific antibodies and N-terminal microsequencing. This research will provide a logical protein chemistry-based approach for assessment of the structure of the reserpine binding site of the vesicle monoamine transporter. This work will provide insight into the mechanism of action of the monoamine transporters and contribute to our understanding of how pharmacological and therapeutic strategies may be devised to treat Parkinsonism, cardiovascular disease, allergy, and disorders of the nervous system.
