Protein kinase C [PKC] is a key regulatory element in synaptic signal transduction. During the previous period of support it was found that alcohols and general anesthetics inhibit PKC with a potency that is a linear function of the octanol/buffer partition coefficient indicating a hydrophobic binding site. The effect follows the Meyer-Overton """"""""rule"""""""" of alcohol and anesthetic action which requires that anesthetic effects be a linear function of hydrophobicity. The measurements were made in the absence of membrane lipids suggesting the site is on the molecule itself. This observation has also been obtained at the single isoform level with purified recombinant PKCalpha. Evidence obtained so far points to the phorbol ester binding site as the site of alcohol interaction. While membrane lipid-associated PKCalpha is also inhibited by short chain alcohols, by contrast, long chain alcohols and general anesthetics potentiate activity. To further understand the effects of alcohols and general anesthetics on PKC and its role in ethanol intoxication and general anesthesia it is proposed to extend and broaden the study to encompass the three major PKC isoforms classes since they possess widely differing properties. Also since PKC has been found to target cytoskeletal elements involved in neurotransmission, we propose to investigate this process following preliminary findings showing ethanol effects. To accomplish this the cDNA encoding the major PKC isoforms that occur in brain, has been obtained and inserted into baculovirus vectors and the PKC expressed in Sf9 cells and purified. It is proposed to investigate the effect of alcohols with respect to activator-and substrate-type differences that have been observed in preliminary experiments. The site of alcohol interaction within PKC and its molecular details will be determined using a series of full length and fusion-protein mutants along with the effects of alcohols on the increased proteolytic sensitivity of activated-PKC that leads to down regulation. Overall the results should contribute to understanding of the mechanism and consequences of ethanol and general anesthetics on synaptic signaling and neurotransmission.
