Research Project
3299856
Modern surgery is entirely dependent upon the use of general anaesthetics, yet how these hazardous drugs act to produce reversible loss of consciousness is still unknown. The long-term objective of this work is to understand how general anaesthetics work at the molecular level, with a view to producing more specific and safer clinical agents. The specific aims of this work and how they will be achieved can be summarized as follows. (1) To determine whether tahe primary target sites in general anaesthesia are proteins or lipids. Selected anesthetics, which have recently been found to differentially affect neuronal ion channels, will be tested for their effects on accepted protein (firefly luciferase) and lipid models of general anaesthesia. Apparent enthalpies (delta H) of anaesthetic binding, which discriminate between protein and lipid sites, will be determined and compared for these model systems as well as for neuronal ion channels and general anaesthesia in animals (tadpoles). The differing predictions of protein and lipid theories of the "cutoff" effect will be tested on animals using large anaesthetics (long-chain n-alcohols) which do not produce general anaesthesia on their own. (2) To determine what factors make certain proteins sensitive to a wide range of general anaesthetics. Enthalpies of binding will be determined on the above systems for anaesthetics having differing hydrogen-bond donor and acceptor characteristics to test the recent finding that anaesthetic targets in animals appear to be good hydrogen-bond acceptors but poor hydrogen-bond donors. X-ray crystallographic studies on an enzyme which has a large anaesthetic-binding pocket will locate the position and binding environment of different anaesthetic molecules (including those of clinical relevance) at atomic resolution. (30 To determine if pressure reversal of general anaesthesia can be explained by pressure and anaesthetics acting at the same molecular sites. The first experiments to show that high pressure (approximately 150 atm) can restore consciousness to anaesthetized animals were only performed because previous studies had shown that anaesthetic inhibition of the luminescence rom bacterial cells could be reversed by pressure, yet these early observations have never been followed up by experiments on the pure bacterial luciferase enzyme responsible for the light-emitting reaction. Pressure reversal of the anaesthetic inhibition of highly purified bacterial luciferase will be investigated using a specially constructed high-pressure rapid-mixing chamber that has been successfully used with firefly luciferase.
