There are two components to this Core, electrophysiology ancUmaging. I. The electrophysiology component of this Core will provide resources to qualitatively screen novel conopeptides to identify their target as well as quantitatively assess the functional activities of conopeptides against known ion channel targets. Assays at three levels will be performed, where each successive level involves decreased scope and increased specificity: 1) Extracellular recording from amphibian and rodent skeletal muscle and peripheral nerves (including motor, sensory, and sympathetic nerves) in isolated tissue preparations will be used to obtain a global assessment of a conopeptide's activity. 2) Whole-cell voltage clamping of dissociated neurons will be used to identify the type of the channel affected by a given conopeptide (e.g., voltage-gated Na vs. K channels). 3) The specific channel isotype targeted by the conopeptide will be pinpointed by examining a conopeptide's effect on cloned channels expressed in Xenopus 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 conopeptide binding and action at the tissue, cellular, and molecular levels by light microscopy. There are three facets to this endeavor. 1) Derivatize conopeptides that have known target-specificities with fluorescent reporter groups. Characterize the adducts'binding affinities, target specificities, and functional properties to see if any of these are altered by the presence of the reporter group. 2) Use of the labeled conopeptides to identify the locations of their binding sites in isolated tissues and in brain and spinal cord slices by confocal fluorescence microscopy. 3) Examine the interactions of conopeptides with their target channels at the molecular level by single molecule imaging using total internal reflection (or evanescent wave) fluorescence microscopy (TIRFM). Here, fluorescently labeled conopeptides will be used in conjunction with Xenopus oocytes expressing cloned channels and subsequently dissociated cells, a) The number of conopeptide molecules bound to a single channel will be determined by counting bleaching-steps. b) For nAChRs that are permeable to Ca++, functional consequence of conopeptide-binding can be concurrently monitored by calcium imaging.
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