To elucidate the mechanisms involved in the inhibition of nicotinic acetylcholine receptors (AcChoRs) by general anesthetics, we will map their sites of binding within the AcChoR. It is our hypothesis that some anesthetics, including barbiturates and certain alcohols, inhibit AchoRs primarily by binding within the structure of the ion channel while others, including steroid anesthetics, interact with regions of the AcChoR distinct from the ion channel, primarily at the protein-lipid interface. We will use biochemical and protein chemistry techniques to identify those sites in terms of the known amino acids sequences of the AcChoR subunits and also to identify the regions of the AcChoR involved in the binding of Substance P, and undecapapeptide that is a potent nicotinic postsynaptic membranes isolated from Torpedo elector organ. The structures of the anesthetic binding sites will be determined by characterizing their effects on the binding of reversible radiolabeled antagonists and by the use of radiolabeled photoaffinity labels that will be covalently incorporated into Anchors. Labeled AcChoR subunits will be isolated and degraded so that the sites of labeling can be determined by N-terminal sequence analysis of isolated peptides.
