Specificity of calcium channels in neuronal signaling

说明(申请人提供)：电压依赖性钙离子通道 跨膜蛋白，它允许钙离子在激活时进入。除了……之外 它们的生电作用，钙离子通道提供了膜之间的关键纽带 去极化和广泛的细胞功能。CA2+的作用通常是 当地的，靠近它的入口处。钙离子的作用也是非常具体的。 通过不同类型的钙离子通道的钙离子内流可以激活不同的 蜂窝信号级联。例如，与L式的钙离子通道相比， 钙离子通过NMDA受体内流激活一条不同的信号通路 基因表达的调控。其丰富多样的胞内 靶点，钙是如何实现特异性并只激活一个子集的？ 神经元中的那些靶点？ 长期目标是了解钙离子的作用和分子机制。 神经元信号传递中的通道。具体地说，在这个提案中，我们将测试 认为钙离子通道和钙离子通道之间存在特定相互作用的假设 某些其他的细胞内蛋白质。这种相互作用具有功能性。 意义。具体目标包括分离和鉴定蛋白质， 它们与钙离子通道相互作用。 利用酵母双杂交系统，我们筛选了一个含有 三种不同的钙通道α1亚基的C末端作为诱饵。177个克隆 已经进行了测序。已经选择了三个克隆进行功能研究。 它们包括动力蛋白复合体的轻链Tctex-1；克隆L157和 N397，两种不同形式的PKC结合蛋白。实验正在进行中 探讨钙离子相互作用的功能意义 渠道和克隆在以下几个方面：(1)差异化 不同类型钙离子通道在神经元中的分布(Tctex-1)；和(2) 通过PKC和/或PKC信号的启动来调节通道活动 层叠(L157和N397)。初步数据表明，两者之间的相互作用 Ca~(2+)通道和我们选择的这些克隆确实具有 意义正如我们所假设的那样。

DESCRIPTION (provided by applicant): Voltage dependent Ca2+ channels are transmembrane proteins, which allow Ca2+ entry upon activation. In addition to their electrogenic role, Ca2+ channels provide a pivotal link between membrane depolarization and a wide range of cellular functions. Ca2+ action is often local and close to its source of entrance. Ca2+ action is also very specific. Ca2+ influx through different types of Ca2+ channels can activate distinct cellular signaling cascades. For instance, compared to L-type Ca2+ channels, Ca2+ influx through NMDA receptors activates a distinct signaling pathway for regulation of gene expression. With its abundant and varied intracellular targets, how is Ca2+ able to achieve specificity and activate only a subset those targets in neurons? The long-term goal is to understand the role and molecular mechanisms of Ca2+ channels in neuronal signaling. Specifically, in this proposal, we will test the hypothesis that specific interactions exist between Ca2+ channels and certain other intracellular proteins. Such interactions are of functional significance. Specific aims include isolation and characterization of proteins, which interact with Ca2+ channels. Using yeast two-hybrid system, we have screened a brain cDNA library with the C-termini of three different Ca2+ channel alpha1-subunits as baits. 177 clones have been sequenced. Three clones have been selected for functional studies. They include tctex-1, a light chain of the dynein complex; clones L157 and N397, two distinct forms of PKC binding proteins. Experiments are in progress to address the functional significance of the interactions between Ca2+ channels and those clones in the following aspects: (1) differential distribution of different types of Ca2+ channels in neurons (tctex-1); and (2) modulation of channel activities by PKC and/or initiation of the PKC signaling cascade (L157 & N397). Preliminary data indicate that the interaction between Ca2+ channels and these clones we selected indeed bears the functional significance as we had hypothesized.