Neurons transmit information by releasing neurotransmitters into the synaptic cleft. Release is triggered by increases in intracellular Ca2+ concentration and is mediated by the fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. While these aspects of synaptic transmission are well established, the molecular mechanism that couples Ca2+ to exocytosis is not known. The synaptic vesicle protein synaptotagmin binds Ca2+ and is essential for rapid and efficient Ca2+-triggered exocytosis. However, little is known concerning the molecular mechanism(s) by which synaptotagmin operates in the release process. The long term goal of our research is to elucidate the biochemical function of synaptotagmin in excitation- secretion coupling. To address this question, three Specific Aims are proposed: first, we will continue to delineate the synaptotagmin signaling pathway by identifying and characterizing synaptotagmin.target protein complexes. These studies include extensions of previously identified synaptotagmin.effector interactions, the characterization of preliminary effectors, and the identification of novel effectors. Second, we will determine whether individual synaptotagmin.effector interactions participate in Ca2+-triggered exocytosis. These studies will involve a detailed analysis of the structural determinants that mediate synaptotagmin.effector assembly. Binding domain data will be used to design peptides that potently and specifically inhibit discrete interactions in vitro. The function of individual interactions will then be determined by assessing the effects of the peptides on Ca2+- triggered exocytosis from semi-intact secretory cells. Third, we will determine the association kinetics of individual Ca2+- dependent synaptotagmin.effector interactions. From this analysis we can determine the temporal order of these interactions and discern which interactions are rapid enough to trigger release. This real time analysis will make us of synaptotagmin inserted into artificial liposomes and represents an initial step in the reconstitution of the molecular machinery that underlies Ca2+-triggered membrane fusion. A better understanding of the mechanisms of neuronal exocytosis will provide a framework for studying how this process is modulated and thus contributes to synaptic plasticity in both normal and pathophysiological states. Finally, defining this mechanism should ultimately provide targets for treatment of diseases in which synaptic transmission is impaired.
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