We have found that the lateral domain structures of lipids in membranes are targets of acute alcohol action, and targets of the adaptive response to chronic ethanol and nitrous oxide exposure. Our colligative thermodynamic framework provides a quantitative description of alcohol and anesthetic action on domain structure in membranes. This project aims to elucidate the factors that determine the action of alcohol, anesthetics, and temperature on the lateral lipid domains in membranes, and the linkage, if any, between 'membrane tolerance' and domain formation. Initially, we propose to identify and characterize domain formation in rat liver microsomal extracts and intact membranes from the chronic ethanol and nitrous oxide paradigms. A quantitative description of alcohol-sensitivity and temperature-sensitivity will be made using partitioning analyses in conjunction with probes of membrane dynamics and calorimetric determinations of enthalpy. The response to alcohol and temperature will be analyzed using the quantitative thermodynamic framework developed in this laboratory. Once the biophysical parameters governing the action of alcohols and anesthetics on domain formation are described, inquiry into the compositional requirements that govern domain formation will be initiated. Liposomes of varied composition will be studied to determine the contributions of headgroup, acyl-chain, sterol, and bound counterion on domain formation. Once the compositional requirements of domain formation have been elucidated for the microsomal systems, other membrane systems will be examined (liver mitochondria, skeletal muscle sarcoplasmic reticulum, synaptosomes, erythrocytes). After the physical basis of domain formation is identified, the chemical origins of the adaptive response that modulates domain formation will be addressed using analytical lipid biochemistry and high resolution nuclear magnetic resonance. Commonalities among the chronic paradigms will provide a method to screen for potentially relevant alterations. These studies will describe the acute and chronic manifestations of ethanol exposure upon membrane lipid topography. Behavioral measures of anesthetic potency at the alcohol 'cutoff' provide a test to distinguish between the predictions of lipid and protein models for anesthesia. Artemia salina will be exposed to varying concentrations of ethanol and aliphatic alcohols. Isobolograms based on phototropism will be constructed to quantitate the effects of the aliphatic alcohols. Protein models for anesthetic action postulate a hydrophobic protein site of circumscribed dimension; consequently, long-chain aliphatic alcohols are deemed nonanesthetic. Our thermodynamic framework for alcohol action on membrane lipids allows for an antagonism of ethanol action. These studies will further define the locus for alcohol action in the brain.
