Each year millions of people undergo general anesthesia so that surgeons can perform life-saving operations. For most patients general anesthesia is safe and effective, however, side effects, such as nausea and vomiting, and complications, such as cardiovascular instability or cognitive dysfunction, increase anesthesia-associated morbidity. Understanding the molecular basis of general anesthetic action may lead to the identification of newer and safer general anesthetics. The long term goal of this project is to elucidate the molecular bases of general anesthesia. Propofol, a widely used intravenous general anesthetic, is the major focus of this project. At concentrations used clinically, propofol interacts with many proteins, however, a major target for its anesthetic effects is the GABA-A receptors. This project will focus on propofol's interactions with GABA-A receptors. In order to understand the molecular basis of propofol's action we must identify the amino acids that form its binding sites, elucidate the conformational changes that it induces, and determine how these conformational changes modulate receptor function. Our ability to accomplish these goals took a quantum leap forward with the recent publication of the homologous acetylcholine binding protein (AChBP) and nicotinic acetylcholine (ACh) receptor structures. These structures provide a solid foundation for structure-based studies of anesthetic interactions with GABA-A receptors. This structural information will guide our experimental design and the interpretation of our results. We propose the following Specific Aims: 1) To identify the residues that lie in close proximity to the propofol binding site near the beta subunit Met286 position and to determine the orientation of propofol in the binding site using sulfhydryl- reactive propofol derivatives. 2) To probe the extent of conformational changes induced by potentiating concentrations of propofol in the M2 segment that lines the ion channel and forms the channel gate. These experiments will serve the dual purposes of verifying the applicability of the ACh-derived structural information to GABA-A receptors and placing structure-function studies of anesthetic interactions with ion channels on a sound structural basis. The successful completion of this project will provide dynamic information on propofol's interactions with GABA-A receptors and new insights into the molecular basis of general anesthetic action. They may provide a rational basis for design of new, safer general anesthetics.
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