The electrophysiology component of this Core will screen conotoxins for their functional activities against known ion channel targets as well as identify possible new targets for novel conotoxins. This will be pursued at three levels, with each succesive level having decreased scope but increased specificity. A) Extracellular recording from skeletal muscle and motor, sensory, and sympathetic nerves in isolated tissue preparations will be used to obtain a global assessment of a toxin's activity. B) Whole-cell voltage clamping of dissociated neurons will be used to focus in on the general nature of the channel affected by the toxin (e.g., Navs K; fast- vs slow-inactivating, etc.). C) The specific channel isotype targeted by the toxin will be pinpointed by examining the toxin's effect on cloned channels expressed in Xenopus oocytes. Levels B and C will also address the mechanism of toxin-action. The histology component of the Core will identify the sites of conotoxin binding in excitable tissues by light microscopy. Toward this end, conopeptides will be labeled with the following reporter groups: (a) fluorophores, for direct viewing by fluorescence microscopy; (b) 125I, for use with autoradiography and viewing by dark field microscopy; and (c) biotin, for use with avidin conjugated to HRP for histochemical staining and avidin conjugated with fluorophores for fluorescence microsocopy. These experiments assessing the locations of the cellular and subcelluar binding sites of toxins will complement the physiological experiments assessing the toxins' effects on target function.
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