Specificity of calcium channels in neuronal signaling

描述（由申请人提供）：电压依赖性Ca 2+通道是 跨膜蛋白，其在激活时允许Ca 2+进入。除了 它们的生电作用，Ca 2+通道提供了膜之间的关键联系， 去极化和广泛的细胞功能。Ca 2+的作用往往是 靠近入口的地方。Ca 2+的作用也非常具体。 通过不同类型的Ca 2+通道的Ca 2+内流可以激活不同的Ca 2+通道。 细胞信号级联。例如，与L型Ca 2+通道相比， 通过NMDA受体的Ca 2+内流激活了不同的信号通路， 基因表达的调控。其丰富多样的细胞内 Ca 2+是如何实现特异性并仅激活一个子集的？ 神经元中的那些靶点 长期目标是了解Ca 2+的作用和分子机制 神经元信号传导的通道。具体而言，在本提案中，我们将测试 Ca 2+通道之间存在特异性相互作用的假设， 其他细胞内蛋白质。这种相互作用具有功能性 意义具体目标包括蛋白质的分离和表征， 与Ca 2+通道相互作用。 利用酵母双杂交系统，我们筛选了一个脑cDNA文库， 三种不同Ca 2+通道α 1-亚基的C-末端作为诱饵。177个克隆 已经被测序。已选择三个克隆进行功能研究。 它们包括tctex-1，动力蛋白复合物的轻链;克隆L157和 N397，两种不同形式的PKC结合蛋白。实验正在进行 为了解决Ca 2+和Ca 2+之间相互作用的功能意义， 通道和这些克隆在以下方面：（1）差异 不同类型的Ca 2+通道在神经元中的分布（tctex-1）;和（2） 通过PKC调节通道活性和/或启动PKC信号传导 级联（L157和N397）。初步数据表明， Ca 2+通道和我们选择的这些克隆确实具有功能性Ca 2+通道， 就像我们假设的那样。

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.