How general anaesthetics produce reversible loss of consciousness is unknown. The long-term objective of this research is to understand the molecular mechanisms underlying general anaesthesia. If the molecular target sites can be characterized, then it should be possible to design and develop new anaethetic agents which are much safer than those currently available. The proposed research is directed towards answering three questions. (1) Are the primary target sites lipid or protein or both? Novel anaesthetic agents, designed to bind differentially to lipid bilayers and proteins, will be used. The protencies of these agents for producing general anaesthesia will be measured in tadpoles and correlated with their binding to and functional effects upon lipid bilayers and a particularly sensitive, highly purified protein (firefly luciferase). (2) Why are some proteins much more sensitive than others to inhibition by a wide range of genral anaesthetics? preliminary studies have identified enzyme forms (i) extremely sensitive to a wide range of anaesthetics (firefly luciferase), (ii) sensitive to some anaesthetics cut not others (bacterial luciferase), and (iii) insensitive to most anaesthetics (a low ATP form of firefly luciferase). In order to account for these differences in selectivity and sensitivity, the anaesthetic-binding pockets of these three enzyme forms will be probed using selected sets of anaesthetic agents which difffer in size, shape, and polarity distribution. (3) Is the inhibition of particularly sensitive proteins by anaesthetics reversed by high pressure? High pressures can restore consciousness to an anaesthetized animal. To investigate a possible molecular basis for this phenomenon, the effects of high pressures (up to 300 atm) on the interactions between anaesthetics and luciferase enzymes will be studied using a specially constructed high-pressure chamber.
