Specificity of calcium channels in neuronal signaling

描述(申请人提供)：电压门控钙通道(VGCC)是跨膜蛋白，允许钙离子在激活时进入。除了它们的生电作用外，VGCC还在膜去极化和广泛的非电活动之间起着至关重要的作用。钙离子通过不同类型的VGCC内流可以激活/调节不同的细胞信号级联反应。VGCC也是各种第二信使调制的目标。磷酸化和去磷酸化是快速调节钙通道活性的重要手段。新的证据表明，大分子信号复合体的形成是钙离子通道作用的特异性以及蛋白激酶和/或磷酸酶快速调节通道活性的机制之一。该项目的长期目标是了解钙通道伙伴蛋白在神经元信号转导中的独特作用。在这一应用中，我们将集中在三个新的钙通道伙伴蛋白，通过酵母双杂交筛选鉴定，它们在形成PKC信号复合体以及将mRNA靶向树突和/或轴突以进行活性依赖的局部蛋白质合成中发挥作用。将使用分子和细胞生物学、生物化学、荧光成像和电生理学的组合方法来检验几种不同的假说。我们将讨论：(1)生理因素，如蛋白激酶的磷酸化，调节PKCE-ENH-N-型钙通道复合体的形成。(2)存在新的钙结合结构域，负责对PKCE-ENH和ENH-N型钙通道之间的相互作用进行不同的调节。(3)具有功能的PKC信号复合体包括一个蛋白磷酸酶PP2ca，它直接与钙通道的C末端结合。(4)一种新的通道伙伴蛋白PQ-46是一种与N-和P/Q型钙通道结合的RNA结合蛋白，它可能参与了mRNAs对树突和/或轴突的活性依赖性靶向。这些结果将阐明通过蛋白质-蛋白质相互作用建立的细胞信号网络是如何实现其特异性的。这项研究的结果将提供大脑中信号转导的分子机制，并将有助于了解学习和记忆等正常神经功能，并为阿尔茨海默病等神经疾病提供原因。

DESCRIPTION (provided by applicant): Voltage-gated Ca2+ channels (VGCCs) are transmembrane proteins, which allow Ca2+ entry upon activation. In addition to their electrogenic role, VGCCs provide a pivotal link between membrane depolarization and a wide range of non-electrical activities. Ca2+ influx through different types of VGCCs can activate/regulate distinct cellular signaling cascades. VGCCs are also targets for modulation by a variety of second messengers. Phosphorylation and dephosphorylation are important means of rapid regulation of the Ca2+ channel activity. Emerging evidence shows that formation of macromolecular signaling complexes is one the of the mechanisms for the specificity of the Ca2+ channel action as well as rapid regulation of the channel activity by protein kinases and/or phosphatases. The long term goal of this project is to understand the unique role of Ca2+ channel partner proteins in neuronal signaling. In this application, we will focus on three novel Ca2+ channel partner proteins, identified via yeast two-hybrid screening, for their roles in formation of a PKC signaling complex as well as in targeting mRNAs to dendrites and/or axons for activity-dependent localized protein synthesis. Several different hypotheses will be tested, using the combined approaches of molecular and cell biology, biochemistry, fluorescent imaging and electrophysiology. We will address: (1) Physiological factors, such as phosphorylation by protein kinases, regulate formation of the PKCe-ENH-N-type Ca2+ channel complex. (2) Novel Ca2+ binding domains exist, responsible for differential regulation of the interactions between PKCe-ENH and ENH-N-type Ca2+ channels. (3) The functional PKC signaling complex includes a protein phosphatase, PP2ca, which binds directly to the C-terminus of Ca2+ channels. (4) A novel channel partner protein, PQ-46, which is an RNA binding protein and binds to N- and P/Q-type Ca2+ channels, may be involved in activity dependent targeting of mRNAs to dendrites and/or axons. The results will shed light on how the cellular signaling network, established via protein-protein interactions, achieves its specificity. Results of this study will provide molecular mechanisms of signal transduction in the brain and will help understand normal neurological functions, such as learning and memory, and provide reasons for neurological disorders, such as Alzheimer's disease.