It is proposed to determine the effects of ethanol on synaptic membrane- associated protein kinase C (PKC). The enzyme becomes associated with the membrane in a calcium- and phosphatidylserine-dependent manner as a result of an increase in intracellular calcium and interaction of diacylglycerol produced by the action of phospholipase C. It then phosphorylates and thereby induces conformational changes in target proteins, such as ion channels, receptors, etc., which lead to various functional changes in these proteins. The functioning of many synaptic membrane proteins modulated by PKC are also affected by ethanol, and there is increasing evidence that some ethanol effects may be directly mediated through an affect on PKC. Preliminary data show that ethanol and higher alcohols inhibit PKC purified from brain, the potency increasing linearly with the membrane/buffer partition coefficient of the alcohol. Although the alcohols fluidized the lipids, it was found that increasing the membrane fluidity by other means led to a potentiation of activity rather than inhibition. This, and the fact that the inhibition by alcohols appeared to be independent of the presence of lipids, implicate a direct interaction of the alcohols with enzyme. To further investigate these effects and to understand the mechanism, a model is proposed which suggests that ethanol may have a dual effect on PKC, both activating (due to fluidication) and inhibiting (by direct interaction). It is proposed to test the possibility that the net effect depends on the ability of the ethanol to fluidize the membrane lipids. the model will be tested by studying the action of ethanol and the higher alcohols using various spectroscopic (fluorescence, NMR and ESR) and other kinetic approaches. The studies will be performed both at the level of the endogenous synaptic membrane-associated PKC and with purified PKC isoenzymes and model lipid bilayers.
