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 depression by altering levels of serotonin, norepinephrine, dopamine, or other neurotransmitters. The strategy of this proposal is based on the rationale that identification of the inhibitor, substrate, and proton translocation sites on monoamine transporters and VMAT-interacting proteins 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. This work will be accomplished in four Specific Aims: (1) identification of the VMAT2 substrate binding site(s) using novel derivatives of amphetamine, dopamine, and the neurotoxin MPP+; (2) identification of the reserpine binding site(s) on VMAT2 using reconstituted pure VMAT2 in artificial liposomes and novel radioiodinated reserpine photolabels; (3) identification of the specific [14C]dicyclohexylcarbodiimide (DCCD)-reactive amino acid in VMAT2 which is involved in proton transport; novel cleavable DCCD-based photolabels to probe the molecular environment of the DCCD-reactive site will be prepared; (4) identification of proteins which interact with VMAT using multiple biochemical and molecular biology techniques. This research will provide a logical protein chemistry-based approach for assessment of the structure and regulation 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.
