STUDY ON THE MECHANISM OF BLOCKADE OF ACETYLCHOLINE RELEASE BY SNAKE PRESYNAPTIC PLA2 NEUROTOXINS ON NERVE TERMINALS O. Rossetto, M. Rigoni, P. Caccin, C. Montecucco Department of Biomedical Sciences, University of Padova, Italy Several animal venoms contain toxins with phospholipase A2 (PLA2) activity. These enzymes hydrolyse the sn-2 ester bond of 1,2-diacyl-3-snphosphoglycerides producing fatty acids and lysophospholipids. Some snake venoms contains presynaptic PLA2 neurotoxins that cause a persistent blockade of neurotransmitter release from nerve terminals. Three subsequent phases can be distinguished at the neuromuscular junction (NMJ) poisoned by PLA2 neurotoxins: a short initial phase with either decreased or unchanged ACh release, is followed by a longer phase of facilitation of ACh release, which then fades into the third phase of complete and irreversible inhibition of neurotransmission. Electron microscopy studies of poisoned NMJ revealed appearance of many clathrincoated W-shaped plasma membrane invaginations, indicating a blockage of endocytosis. Therefore PLA2 neurotoxins both promote fusion of small synaptic vesicles (SSVs) with the presynaptic membrane and inhibit their retrieval, thus causing release of ACh, depletion of vesicles and enlargement of nerve terminals. On the basis of these data we are investigating whether PLA2 neurotoxins block nerve terminals entering the lumen of synaptic vesicles and hydrolyse phopholipids of the inner leaflet of the membrane. The suggested entry of PLA2 neurotoxins inside SSVs would account for the finding that electrical activity of the nerve terminals actively promotes intoxication. In order to test our hypothesis we are studying the intracellular localization of two snake PLA2 neurotoxins (beta-bungarotoxin and taipoxin) on mouse phrenic nerve-hemidiaphragm NMJ intoxicated with these toxins. If this working hypothesis proves true, the presynaptic PLA2 neurotoxins could be employed as tools to investigate specific aspects of acetylcholine release and of vesicles fusion and recycling.
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