The long term objective of our research is to provide a molecular description for the mechanism of action of volatile anesthetics (VA) on membrane proteins whose functional impairment might be related to anesthesia. Our approach is based on the hypothesis that VA interfere with normal Ca2+ homeostasis in the cell. The proposal takes advantage of the availability of a purified, well defined system, the red blood cell (RBC) membrane Ca2+-ATPase that allows us to test certain hypotheses and get clear-cut answers. Significant inhibition of the Ca2+-ATPase activity by VA has been demonstrated that correlates well with the clinical potency of VA. Our hypothesis is that the VA act on the enzyme protein, either directly or indirectly, to change the equilibrium between enzyme monomers and oligomers and to interfere with binding of the regulatory protein, calmodulin. A series of seven halogenated VA, including VA commonly used in clinical practice as well as experimental VA considered for clinical use, will be tested in systematic studies. Their effects will be compared to other selectively chosen general anesthetics, including aliphatic n-alcohols and paraffins. Studies will be performed on purified enzyme as well as on enzyme reconstituted in RBC phospholipids and on intact RBC. Kinetics of the enzymatic reaction will be measured under steady state conditions, and if necessary in the transient state (millisecond time scale) to determine which step in the reaction cycle is affected. After tagging the enzyme and its regulatory protein, calmodulin, with fluorophores the effect of anesthetics on the extent of enzyme oligomerization will be determined by fluorescence energy transfer and the binding of calmodulin to enzyme monomers will be followed through fluorescence intensity measurements. Changes in lipid fluidity will be determined by fluorescence anisotropy and correlated to changes in intracellular free Ca2+ concentrations measured by 19F NMR techniques. Red blood cell is a classical model cell and after explaining how the Ca2+- ATPase and Ca2+ homeostasis are affected by VA in this relatively simple system the findings could be applied to more complex systems, such as brain or muscle. Understanding of the site and mechanism of the VA action will allow future prevention of anesthesia-induced complications.
