The molecular mechanisms of neurotransmitter storage and release in the mammalian nervous system are poorly understood. In research to elucidate these mechanisms the major proteins of mammalian synaptic vesicles have been identified and characterized. The functions of these proteins are not known. Experiments designed to identify ion transport and structural functions of major synaptic vesicle proteins and to generate antibodies to these proteins are proposed. I) An ATP-dependent proton pump is required for uptake of neurotransmitters by synaptic vesicles. Enzymological and solute transport experiments are planned to show that a multisubunit protein complex recently identified on brain synaptic vesicles and composed of several of the major proteins is, in fact, the proton pump that drives neurotransmitter uptake. The question of whether one major synaptic vesicle protein, p65, is a component of the putative pump will be investigated by cross-linking. II) Interactions of synaptic vesicle proteins with the presynaptic cytoskeleton and plasma membrane are postulated to control neurotransmitter release. With synaptic vesicles of >90% purity from rat brain, prepared by a method I have developed recently, such interactions can now be studied directly. Two approaches will be pursued: (1) Saturable, high-affinity interactions of synaptic vesicles with individual, purified proteins or with larger presynaptic components will be studied with a solid-phase screening assay, velocity sedimentation, affinity chromatography using immobilized synaptic vesicles, and gel overlay techniques. The synaptic vesicle proteins involved in such interactions of synaptic vesicles with individual, purified proteins or with larger presynaptic components will be studied with a solid-phase screening assay, velocity sedimentation, affinity chromatography using immobilized synaptic vesicles, and gel overlay techniques. The synaptic vesicle proteins involved in such interactions will be identified by affinity labeling, cross-linking, or gel overlay methods. (2) Tubulin, which can interact with several cytoskeletal elements, is one of the major synaptic vesicle proteins. interactions of tubulin with other proteins in intact synaptic vesicles will be identified by cross-linking and binding studies, the isotypic composition of vesicular tubulin characterized electrophoretically and immunochemically, the distribution of tubulin in the synaptic vesicle population determined by immuno-precipitation, and a large protein complex containing tubulin characterized. III) Antibodies to synaptic vesicle proteins. Immunological reagents for most of the major proteins of mammalian synaptic vesicles are currently unavailable. Recent success in raising polyclonal antisera to many of these proteins in my laboratory indicates that isolation of monoclonal antibodies is now feasible. In addition to raising such antibodies, a general method to screen for monoclonal antibodies to epitopes whose distribution is restricted to particular synaptic vesicle subclasses will be utilized. The work summarized here will provide knowledge as well as immunological tools that should be helpful in understanding the cellular and molecular mechanisms of neurotransmission in mammals. ultimately these studies may contribute to the development of new therapeutic approaches to modulate synaptic function.
