Characterization of botulinum neurotoxin subatrates and binding proteins in mouse neuromuscular tissue
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Botulinum toxin is the most lethal toxin known. Its significance as a biological agent is paradoxical since on the one hand it can cause the fatal disease known as botulism; while at the same time it is the treatment of choice for a variety of medical disorders ranging from painful dystonias to facial wrinkles. Of a more serious and timely concern, however, is its potential threat as an agent of biological warfare due to its ease of production, extremely high potency and specificity of action at the neuromuscular junction. Moreover, for scientists, botulinum toxin is a valuable tool in the dissection of transport pathways and transmitter release mechanisms. Early studies established a model of the mechanism of toxin action at the neuromuscular junction, the target organ of botulinum toxin. However, several questions such as the exact nature of its intracellular action and the identity of its membrane receptors remained unanswered. To address these questions, many elegant studies have been conducted using cell culture, protein expression systems or brain synaptosomes; however, it should not be assumed that the results would be similar in target tissues. Mouse neuromuscular junction was utilized in the studies herein because this ex vivo hemidiaphragm preparation incorporates all the processes normally occurring during intoxication with botulinum neurotoxin. Using electrophysiology, electrophoresis and immunological methods, I identified the substrates of botulinum toxin serotypes A, B, C, and D in the mouse hemidiaphragm. Further, I related paralysis caused by toxin to the proteolysis of toxin substrates, and utilized pharmacologic agents to confirm that proteolysis was directly mediated by the multi-step mechanism of toxin action. Finally, I demonstrated that the vesicular proteins synaptotagmin I and II are toxin-binding proteins for serotype A and B, respectively from this preparation. The functional relevance of these binding proteins in this preparation was also investigated.