It is our hypothesis that membrane structural fluctuations play an important role in the modulation of membrane-protein function. Assuming that the site of anesthetic action is the lipid matrix of the biological membrane, we propose that the initial event in anesthesia is alteration of such fluctuations. This hypothesis will be tested using the gel to liquid- crystalline transition and compositional-based phase separations in single bilayer vesicles as models for such fluctuations. The effects of general and local anesthetics on the equilibrium properties of these transitions will be investigated using calorimetric and spectroscopic techniques. Particular emphasis will be placed on quantitative evaluation of the anesthetic-induced changes in the melting temperature and shape of the heat capacity function associated with the thermotropic transitions. The former parameter is a measure of the differential solubility of the anesthetic in the gel and liquid-crystalline states; the latter parameter is related to the details (e.g. cluster size) of the distribution of distinct molecular species of the lipids. The dynamics of the transitions will also be investigated using a novel, volume perturbation, dynamic calorimeter. An important aspect of this study is the investigation of how anesthetics alter the vesicle-surface activation of the enzyme phospholipase A2 which appears to be strongly coupled to lipid structural fluctuations. These structural fluctuations depend upon temperature, lipid composition and reaction products which can induce spontaneous activation of the enzyme. This is particularly important since all membrane constituents can potentially alter membrane fluctuations. The proposed experiments will provide a library of thermodynamic and kinetic information related to define lipid-enzyme system, allowing construction of specific quantitative statements describing anesthetic action in terms of its influence on membrane structure and membrane-mediated protein function. Reaction microcalorimetry, differential scanning calorimetry, fluorescence spectroscopy, and pH stat techniques will be used in this investigation. This laboratory possesses unusual expertise in these techniques, which will be complemented by other procedures such as electron microscopy and nuclear magnetic resonance. The experimental work will be supplemented by computer simulation (e.g. Monte Carlo calculations) of the models describing the composite of the experimental results.
Showing the most recent 10 out of 27 publications
