The accumulation of catecholamines and serotonin in synaptic vesicles of the CNS is mediated by a transport system (porter) located in the vesicle membrane. This system is the primary target for several pharmacological agents that have been used extensively in research and are of considerable clinical interest. The long- term objectives of the proposed research include identification, reconstitution and characterization of the molecular entities directly involved in transport. Of particular interest are the subunit structure of the porter, and localization and orientation of binding sites for inhibitors such as reserpine and tetrabenazine. These two agents appear to represent distinct classes of inhibitors that may interact with different subunits of the porter complex in order to achieve these goals photoaffinity analogs of tetrabenazine, reserpine and substrates will be used to identify relevant components of the complex ln synaptic vesicles from beef brain, and labeled components will be characterized with respect to molecular weight isoelectric point, and glycoprotein content by lectin affinity chromatography. The effect of membrane energization on photoaffinity labeling will also be assessed. The respective binding components will be solubilized and purified by conventional biochemical methods, including affinity chromatography using inhibitor binding or photoaffinity labeling to follow activity. The ability of purified components to bind substrates and inhibitors will be compared, and the pH dependence of such binding assessed. The type of inhibition exerted by various agents on dopamine transport and reserpine and dihydrotetrabenazine binding to purified components will be examined. In addition to a better understanding of the structure and organization of the porter, the proposed work should yield information useful in the design of new catecholamine depleting therapeutic agents with potential utility in clinical situations that require a reduction of catecholamine available for release to the synapse.
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