Neurotransmitters are stored in synaptic vesicles so that their release may be regulated by neural activity. For classical transmitters, vesicular storage requires transport from the cytoplasm, where many are synthesized or appear after reuptake from the synapse. However, little is known about the molecular basis for vesicular transport, its potential for regulation or its role in synaptic transmission. The long-term objectives of this proposal are to understand how the transport of neurotransmitters into synaptic vesicles influences the processing of neural information and contributes to human neuropsychiatric disease. The proposal focuses on the vesicular transport of monoamines in light of their role in mental illness, drug abuse and Parkinson's disease. We originally used the neurotoxin MPP+ to isolate the first cDNA for a vesicular neurotransmitter transporter, a vesicular transporter for monoamines. The similarity of the predicted protein sequence to bacterial antibiotic resistance proteins supported a role in protecting against neural degeneration such as the form that occurs in Parkinson's disease. The sequence also defined a novel mammalian gene family and we have since isolated the cDNAs for a second vesicular monoamine transporter and a putative vesicular transporter for acetylcholine. The strategy of the proposal is to determine the mechanism, regulation and cell biology of the vesicular transporters by expression of the cloned cDNAs in heterologous systems, then extrapolate this information to their role in behavior and disease using transgenic models and human genetics. The first specific aim addresses the structural basis for transport activity, focusing on substrate recognition, the mechanism of active transport, efflux and the interaction with drugs. Second, we will characterize the regulation of vesicular amine transport by phosphorylation. Third, we will study the intracellular trafficking of the vesicular transporters to understand the mechanisms that determine the sites of transmitter storage. Fourth, we will target disruption of the gene encoding vesicular amine transport int he brain and assess the role of its inheritance in familial Parkinson's disease. Last, we will extend our study to the related transporter for acetylcholine and the unrelated synaptic vesicle proteoglycan SV2 which shows strong homology to nutrient transporters but whose function in synaptic transmission remains unknown. Correlation of the molecular and cellular analysis with the biological models and genetic studies will then indicate the role of vesicular neurotransmitter transport in information processing, behavior and human neuropsychiatric disease.
