Some 25 million patients are given general anesthesia each year in the USA using agents which have a very low therapeutic index. Without a better understanding of the mechanism of action of general anesthesia will be impossible to design safer agents. Current evidence indicates that anesthetics affect synaptic transmission and are active on a super-family of ligand gated channels which include the GABA/A (GABAAR) and the nicotinic acetylcholine receptors (nAcChoR). The latter has the unique advantage of being obtainable in milligram quantities from the electroplax of Torpedo (in a form which is highly homologous with its mammalian counterpart) making the proposed experiments feasible. The overall hypothesis is that there are several different sites for general anesthetics on the nAcChoR, and that each site's affinity varies with the nAcChoR's conformational state. We have provided strong evidence that general anesthetics act at a site near the channel as well as the lipid- protein interface of nAcChoRs. We will specifically focus on anesthetic interactions with these sites in the resting and open states. Derivatives of aliphatic and aromatic general anesthetics which can be photo-activated will be used to establish which sites are responsible for inhibition by comparing the rate at which these agents inhibit the resting and open states with the rate that they photo-label sites in the channel and the lipid-protein interface. The kinetics of photo-labeling will be determined using a rapid mixing device coupled with a novel rapid (1<1 ms) freeze- clamping apparatus which stops the reaction, allowing a subsequent prolonged, high-yield photo-labeling step in the frozen, non-reactive, state. The sites of photo-incorporation in the amino acid sequence will be determined. In addition, the hypothesis that cholesterol-receptor binding sites at the lipid-protein interface modulate heterotropic allosteric interactions between general anesthetics and agonists will be tested in reconstituted nAcChoR and GABAAR. The nAcChoR work is closely related to that in Project II & III, and makes use of the Synthetic & Protein Chemistry Cores. The GABAAR work collaborates with Project IV.
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