PKC'S ROLE IN LEARNING

这项提议的总体目标是确定分子和 一系列钙和磷脂的生化机制- 依赖蛋白激酶(PKC)参与记忆相关的改变 裸目动物眼内B型光感受器的神经兴奋性 粗角软体动物。蛋白激酶C同工酶的氨基酸序列 将通过对cDNA和基因组DNA的核苷酸测序来推断 克隆人。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NAY表达水平的变化 这些通过学习而产生的成绩单。重组PKC同工酶，AS 以及从神经系统中提纯的正宗蛋白质，将是 具有生化特征的。的几个翻译后修改 可能通过学习而产生的PKC，并增加 将对酶进行评估。这些包括：1)催化剂的产生 限制性蛋白水解法的PKC片段，2)膜插入形式的PKC 其在构成上是活性的，或3)PKC的磷酸化形式，具有 活跃度增加。针对不同基因的单抗和多克隆抗体 PKC同工酶将被生成并用于绘制 同工酶，以及在功能研究中。学习产生的增长 B型细胞膜内的PKC浓度将进行现场研究 使用激光共聚焦扫描光学显微镜。电生理学 实验将确定PKC在K通道活动中的减少 在B型光感受器中反映离子的直接磷酸化 通道复合体，或者改为通过最近描述的推定的 G蛋白。这些研究将有助于我们理解 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.