PKC'S ROLE IN LEARNING

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The general goal of this proposal is to determine the molecular and biochemical mechanisms by which a family of calcium- and phospholipid- dependent protein kinases (PKC) contribute to memory-related changes in neural excitability of Type B photoreceptors in the eyes of the nudibranch mollusc Hermissenda crassicornis. The amino acid sequences of PKC isozymes will be deduced through nucleotide sequencing of cDNA and genomic DNA clones. This sequence information will be used to construct oligonucleotide probes for in situ hybridization localization of mRNA transcripts for the different isozymes and nay changes in expression levels of these transcripts produced by learning. Recombinant PKC isozymes, as well as authentic proteins purified from the nervous system, will be biochemically characterized. Several post-translational modifications of PKC that might be produced by learning and increase the activity of the enzyme will be assessed. These include the generation of: 1) a catalytic fragment of PKC by limited proteolysis, 2) a membrane-inserted form of PKC which is constitutively active, or 3) a phosphorylated form of PKC with increased activity. Monoclonal and polyclonal antibodies against different PKC isozymes will be generated and used to map the distribution of the isozymes, as well as in functional studies. Learning-produced increases in PKC concentration within Type B cell membranes will be studied in situ using laser confocal scanning optical microscopy. Electrophysiological experiments will determine whether PKC's reductions in K channel activities in Type B photoreceptors reflect a direct phosphorylation of the ion channel complex, or are instead mediated via a recently described putative G-protein. These studies will contribute to our understanding of the molecular bases of a simple form of associative learning and memory.