Catecholamines and serotonin are concentrated in synaptic vesicles and chromaffin granules prior to secretion by a process that is dependent on the presence of a transmembrane electrochemical proton gradient, positive and acidic inside. The proposed research will deal with identification, purification, reconstitution and characterization of the molecular entities directly involved in such transport (porter). The binding of radiolabeled reserpine and dihydrotetrabenazine, both potent competitive inhibitor of transport, to synaptic vesicles from bovine striatum and bovine chromaffin granules will be characterized with respect to the effects of energization, and the potencies of natural substrates as inhibitors of binding to the energized and unenergized transporter. Binding to subsynaptic fractions from various anatomical regions of the rat CNS will also be assessed, as will the effects of nigrostriatal 6-OH-dopamine lesions. Radiolabeled photoaffinity analogs of inhibitors will be prepared and used to identify the porter in granules and vesicles, and labeled components will be characterized electrophoretically with respect to subunit molecular weight and isoelectric point, and glycoprotein content by lectin affinity chromatography. Using inhibitor binding, or a tracer of photoaffinity labeled material, the porter will be purified and transport activity reconstituted in artificial phospholipid vesicles. The size, orientation and subunit structure of the porter complex in situ will be determined using radiation inactivation/target size analysis, reversible crosslinking, hydrophobic membrane probes, and surface probes in conjuction with inhibitor binding and photoaffinity analogs. Use of similar techniques with the purified, reconstituted porter will be used to assess the effects of energization, substrates and inhibitors on the structure and conformation of the porter. The effect of varying lipid composition on transport and inhibitor binding will be determined and passive uptake and release of substrate by porter-liposomes will be characterized. The hypothesis that lithium indirectly antagonizes the therapeutic catecholamine-depleting effects of reserpine and tetrabenazine by altering phosphoinositide metabolism will be tested by pretreating rats with lithium prior to preparation of synaptic vesicles, and by the appropriate modification of lipid composition in purified reconstituted porter/liposomes.
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