PKC'S ROLE IN LEARNING

本提案的总体目标是确定分子和 钙和磷脂家族的生化机制 依赖性蛋白激酶（PKC）有助于记忆相关的变化， 裸鳃类眼B型光感受器的神经兴奋性 软体动物厚角赫米森达 PKC同工酶的氨基酸序列 将通过cDNA和基因组DNA的核苷酸序列推断 克隆 该序列信息将用于构建 用于mRNA原位杂交定位的寡核苷酸探针 不同同工酶的转录本和表达水平的变化 of these transcripts转录produced生产by learning学习. 重组PKC同工酶，如 以及从神经系统中纯化出来的真正的蛋白质， 生物化学特征 几种翻译后修饰 PKC可能是由学习产生的，并增加细胞的活性。 将对酶进行评估。 这些包括产生：1）催化剂， 通过有限的蛋白水解作用，2）PKC的膜插入形式 其是组成型活性的，或3）PKC的磷酸化形式， 活动增加。 针对不同抗体的单克隆和多克隆抗体 将产生PKC同工酶并用于绘制 同工酶，以及功能研究。 学习带来的增长 将原位研究B型细胞膜内的PKC浓度 使用激光共聚焦扫描光学显微镜。 电生理 实验将确定是否PKC的减少K通道活动， 在B型光感受器中， 通道复合物，或者相反地通过最近描述的推定的 G蛋白 这些研究将有助于我们了解 简单形式的联想学习和记忆的分子基础。

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.