Role of calmodulin kinases on the regulation of Ca^<2+> signaling of central nervous systems

神经元钙/钙调素依赖的蛋白激酶II(CaM)调节重要的神经功能，包括神经递质的合成和释放、离子通道活性的调节、突触的可塑性和基因表达。本研究得到以下结果：1.在神经细胞中引入CaM激酶II，可刺激突起突起生长；2.在突触后密度(PSD)中发现有28种以上的蛋白底物参与突触生长；2.在突触后密度(PSD)中发现有28种以上的蛋白底物，几乎所有这些蛋白都被蛋白质测序和质谱学鉴定；4.观察培养细胞在神经分化过程中的发育变化，发现CaM激酶II在神经元分化过程中上调：5.在大鼠脑组织发育过程中，δ亚型的剪接模式发生了明显的变化，在培养的神经细胞分化过程中也发生了明显的变化，表明在神经分化过程中，δ亚型的剪接异构体也有选择性表达；6.利用β特异性插入的缺失突变体研究α和β的酶特性差异，发现β特异性插入βCaM激酶II在细胞内的分布中起重要作用；7.克隆了编码大鼠钙调素依赖的蛋白激酶IIβ亚型的基因，并确定了其结构和外显子-内含子组织；8.用神经细胞和非神经元细胞分析该基因的启动子活性时，在该基因的5‘-上游区域发现了神经细胞类型特异性启动子活性。

Neuronal Ca^<2+> /calmodulin-dependent protein kinase II (CaM kinase II) regulates important neuronal functions, including neurotransmitter synthesis and neurotransmitter release, modulation of ion channel activity, synaptic plasticity, and gene expression. The following results were obtained in this study; 1. when CaM kinase II was introduced in neuronal cells, neurite outgrowth was stimulated, indicating that CaM kinase II played a role stimulation of neurite outgrowth; 2. Ca^<2+> independent activity of the kinase autophosphorylated at Thr-286 involved in neurite outgrowth; 3. more than 28 protein substrates of CaM kinase II were found in the postsynaptic density (PSD), and almost all of these proteins were identified by protein sequencing and mass spectrometry; 4. when the developmental change of the kinase was investigated during the neural differentiation of cultured cells, it was found that CaM kinase II was upregulated during the neuronal differentiation; 5. cell type distinctive changes of splicing pattern of δ isoform were found not only during development of rat brain tissue, but also during differentiation of cultured neuronal cells, indicating that alternatively spliced variants of δ isoform of CaM kinase II were expressed during neural differentiation; 6. when we used the deletion mutants for β-specific insertions to explore the difference between α and β CaM kinase II in enzymatic properties, it was found that β specific insertion of β CaM kinase II played an important role in the cellular distribution of the kinase; 7 The gene encoding the β isoform of rat Ca^<2+>/calmodulin-dependent protein kinase II was cloned, and its structure and exon-intron organization were determined; 8. When the promoter activity of the gene was analyzed using nueronal and non-neuronal cells, neuronal cell type-specific promoter activity was found in the 5'-upstream region of the gene.