Locating General Anesthetic Sites in Acetylcholine Receptors We wish to identify the sites of binding of representative general anesthetics when they are acting as inhibitors of nicotinic acetylcholine receptors (AcChoRs). The anesthetics to be studied include steroids, which are likely to act at the protein-lipid interface, alcohols and barbiturates, that may act within the ion channel domain, and halothane, a volatile anesthetic that may interact with novel regions of the AcChoR that are important for the gating of the ion channel by agonists. Mapping studies will be carried out with AcChoRs in nicotinic postsynaptic membranes isolated from Torpedo electric organ. Radio-labeled, photo- activatable anesthetics will be covalently incorporated into AcChoRs and labeled AcChor subunits will be isolated and degraded so that the sites of labeling can be determined by N-terminal protein sequence analysis of the labeled peptides. These structural studies provide a definition of the amino acids and regions of the AcChor that tare in contact with the anesthetics. but they do not assess the functional importance of these sites of contact. To address this for steroid antagonists, we will characterize interactions of progesterone and progesterone analogs with wild type and mutant AcChoRs expressed in Xenopus oocytes to identify structural determinants important for drug potency and to determine how mutations within different AcChoR functional domains alter drug potency.
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