There are two components to this Core, electrophysiology ancUmaging.I. The electrophysiology component of this Core will provide resources to qualitatively screen novelconopeptides to identify their target as well as quantitatively assess the functional activities of conopeptidesagainst known ion channel targets. Assays at three levels will be performed, where each successive levelinvolves decreased scope and increased specificity: 1) Extracellular recording from amphibian and rodentskeletal muscle and peripheral nerves (including motor, sensory, and sympathetic nerves) in isolated tissuepreparations will be used to obtain a global assessment of a conopeptide's activity. 2) Whole-cell voltageclamping of dissociated neurons will be used to identify the type of the channel affected by a givenconopeptide (e.g., voltage-gated Na vs. K channels). 3) The specific channel isotype targeted by theconopeptide will be pinpointed by examining a conopeptide's effect on cloned channels expressed inXenopus oocytes. Levels 2 and 3 will also allow the mechanism of conopeptide-action to be addressed.II. The imaging component of the Core will be used to identify and investigate the sites of conopeptidebinding and action at the tissue, cellular, and molecular levels by light microscopy. There are three facets tothis endeavor. 1) Derivatize conopeptides that have known target-specificities with fluorescent reportergroups. Characterize the adducts' binding affinities, target specificities, and functional properties to see ifany of these are altered by the presence of the reporter group. 2) Use of the labeled conopeptides to identifythe locations of their binding sites in isolated tissues and in brain and spinal cord slices by confocalfluorescence microscopy. 3) Examine the interactions of conopeptides with their target channels at themolecular level by single molecule imaging using total internal reflection (or evanescent wave) fluorescencemicroscopy (TIRFM). Here, fluorescently labeled conopeptides will be used in conjunction with Xenopusoocytes expressing cloned channels and subsequently dissociated cells, a) The number of conopeptidemolecules bound to a single channel will be determined by counting bleaching-steps. b) For nAChRs thatare permeable to Ca++, functional consequence of conopeptide-binding can be concurrently monitored bycalcium imaging.
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