The broad goal of this proposal is to better understand molecular mechanisms which regulate neurotransmitter release. The focus is on the proteins which interact with alpha-latrotoxin, a neurotoxic component of Black Widow Spider venom. Alpha-Latrotoxin is the strongest known stimulator of spontaneous neurotransmitter release. In addition, a long term effect of alpha- latrotoxin application is degeneration of nerve terminal. The toxin's action can be explained in part by calcium fluxes through the cation channels generated by the toxin in the presynaptic membrane. However, alpha-latrotoxin also stimulates neurotransmitter release by an as yet unknown mechanism in the absence of extracellular Ca2+ and without a noticeable increase in intracellular Ca2+ concentration. Alpha- latrotoxin binds in a calcium-dependent manner to neurexin Ia, a synapse- specific cytoplasmic membrane protein. Interaction of neurexin-Ia with synaptotagmin, a calcium sensor in the nerve terminal, may be important for alpha-latrotoxin action. In addition to neurexin-Ia preliminary studies identified another high-affinity alpha-latrotoxin receptor. It binds alpha-latrotoxin in the absence of Ca2+ and thus may account for calcium-independent alpha-latrotoxin effects. The synapse-specific location of alpha-latrotoxin receptors and their potential involvement in neurotransmitter release makes these proteins an attractive target for detailed structural and functional studies. Since alpha-latrotoxin can elicit neurotransmitter release without calcium signaling, the calcium-independent alpha-latrotoxin receptor may have a role in regulating a calcium sensor or the components of the docking- fusion machinery involved in neurosecretion. Understanding these mechanisms of neurotransmitter release may provide insight into neurodegenerative and neuromuscular disease. To study the molecular mechanism of alpha-latrotoxin action, calcium- independent alpha-latrotoxin receptors will be purified and molecularly cloned. The information and tools generated by these experiments will be used to address the physiological role of this receptor. The effect of transfection of calcium- independent alpha-latrotoxin receptor on neurotransmitter release will be analyzed in PC12, neurosecretory cell line. The applicants will study the interactions of calcium-independent alpha-latrotoxin receptor(s) with synaptotagmin, syntaxin, and other proteins involved in synaptic transmission process, using a variety of biochemical techniques.
