The proposed research is aimed to investigate the actions of ethanol in vitro and in vivo on neuronal plasma membrane in the brain. Specifically, we will study membrane binding of calmodulin and calmodulin-dependent membrane functions in response to ethanol. Supported by data from preliminary studies, we hypothesize the ethanol changes membrane conformation, leading to changes in membrane binding of calmodulin, and a cascade of membrane events mediated by Ca-calmodulin. Using synaptosomal plasma membrane (SPM) isolated from rat cerebral cortex, five studies are proposed to test this working hypothesis. (1) The first study is to characterize the inhibitory effect of ethanol in vitro on calmodulim binding to the SPM. The changes of calmodulin binding will be correlated with changes in lipid fluidity of the membrane. This study will provide ground work for the later studies. (2) The second study will determine the effects of ethanol on calmodulin-binding proteins in SPM. These binding proteins will be identified by polyacrylamide gel electrophoresis. (3) The third study is to determine by radioimmunoassay the content of calmodulin in the isolated SPM following acute and chronic administration of ethanol. The purpose is to determine if the in vitro effect of ethanol on calmodulin binding is reflected in vivo. (4) The fourth study is to relate the changes in calmodulin binding to changes in the activity of Ca-pump ATPase in the SPM (the calmodulin-dependent Ca-activated ATPase on the plasma membrane). (5) The fifth study is to determine if the changes in calmodulin binding would lead to parallel changes in Ca- calmodulin-dependent phosphorylation of endogenous proteins in the SPM. Thus, the first three studies are aimed at membrane binding of calmodulin, and the last two studies are to examine two membrane functions that are known to be calmodulin-dependent. In all these studies, the SPM from animals with and without chronic ethanol treatment will be compared. The overall objective is to understand the chain of events from changes in membrane conformation to changes in calmodulin- dependent membrane functions in brain. Calmodulin is a ubiquitous Ca-regulating protein present in all types of eukaryotic cells. Thus, the basic information derived from the research on the brain could also be useful as a background in investigating pathogenetic events underlying alcoholism in other cell types such as the liver.
