The aim of this application is the elucidation of the roles of potential key elements in the mechanism of membrane fusion as it occurs in neurosecretory processes. This will be accomplished by specific experiments designed to elucidate the role of the synaptic vesicle protein, synaptotagmin (p65) in neurotransmitter release. Synaptotagmin's role in neurotransmitter release is suggested by 1) its ability to bridge membranes evidenced by red blood cell agglutination; 3) its structure which contains two repeats that have sequence homology to the regulatory region of protein kinase C, a region of PKC involved in calcium dependent membrane interaction; and 4) its calcium dependent interaction with the presynaptic receptor for latrotoxin, a toxin that causes massive exocytosis in synaptic vesicles. Two broad sets of experiments will be performed in order to test the hypothesis that the lipid binding activity of synaptotagmin and calcium dependent interaction of synaptotagmin with the latrotoxin receptor form the basis of docking and/or fusion of synaptic vesicles to the presynaptic membrane. The first set of experiments focus on the identification of the domain of synaptotagmin responsible for calcium dependent interaction with the latrotoxin receptor and on determination of the conditions and regulation of this interaction. The second set involves the use of transfection of synaptotagmin into the neurosecretory cell line, PC12, as a system to study neurotransmitter release. In this system, the membrane traffic of native and recombinant synaptotagmin will be outlined with species specific antibodies. The correlation of extracellular exposure of synaptotagmin epitopes with neurotransmitter release will be determined. Finally the ability of transfection of native or mutated synaptotagmin to enhance or inhibit neurotransmitter release will be tested. Because neurotransmitter release is the pivotal step in communication between neurons, the machinery for release is a potential site for natural or therapeutic modification of nervous system function.
