Calcium Channels, Calmodulin and Nuclear CREB Signaling

 描述(申请人提供)：激发-转录(E-T)偶联是将细胞的电或化学激活转化为传递到细胞核的信号的过程。通过这种方式，基因的表达可以以一种依赖于活性的方式进行调节。神经元重塑被认为对神经元发育、学习记忆和药物成瘾过程中的长期适应性变化是必要的和重要的。E-T偶联最详细的例子是通过Ser133处的磷酸化向转录因子CREB(cAMP反应元件结合)蛋白发送钙信号。电压门控钙通道作为钙离子内流的重要来源，其生物物理和生化特性已被广泛研究。有趣的是，在E-T偶联中，似乎通过不同的钙通道的钙内流可以使不同的信号通路进入细胞核。例如，CaV1(也称为L型)通道比CaV2通道具有很大的优势，尽管CaV1通道只贡献了神经元整体钙进入的一小部分。我们最近的细胞论文发现，这种效力上的差异可以用两类钙通道使用局部和全局钙信号的不同来解释。然而，CaV1通道纳米结构域优势的“专线”还不清楚。现在我们准备提供一个关键问题的详细描述：是什么将长距离信号从CaV1锚定的信号复合体携带到细胞核？我们有一个答案：Ca2/CaM到细胞核的转运依赖于一个共转运体，我们现在认为它是γCaMKII。这种航天飞机收集细胞质中的钙/钙调素，将其隔离在CaV1通道上，然后在核定位信号的控制下传输到细胞核。这种信号机制依赖于γCaMKII、βCaMKII和CaN，这三种信号分子工作在CaV1纳米结构域中，也与多种神经精神疾病有关。这项建议侧重于了解γCaMKII/CaM易位的细胞机制，并提出了三个具体目标。(1)确定CaV1活性与核CREB磷酸化和Cre依赖的转录相关的钙信号机制的动力学。我们将实时跟踪γCaMKII的易位，并评估通过这种穿梭机制运送到细胞核的钙/钙调素的影响。(2)我们将使用基因结构来操纵CREBCaMKII途径，以确定γ磷酸化所需的分子成分。我们将在途径的关键步骤改变涉及CaM、βCaMKII、CaN和PP2A的结合相互作用和酶作用。(3)了解CaV1依赖的CaM在新皮质神经元中的穿梭，明确纳米结构域钙信号和电压门控构象信号在E-T偶联中的不同作用。更清楚地了解CaV1通道和CREB信号之间的联系将对理解基因表达的变化如何改变神经网络中神经元的功能有有利的影响。因此，这项研究既与基础细胞相关，也与基础细胞相关 生物和疾病状态的多样性，如成瘾、自闭症和其他神经精神疾病。

 DESCRIPTION (provided by applicant): Excitation-transcription (E-T) coupling is a process that converts the electrical or chemical activation of a cell to a signal conveyed to the nucleus. n this way, the expression of genes can be modulated in an activity-dependent manner. The neuronal remodeling that results is recognized to be necessary and important for long-term adaptive changes during neuronal development, learning and memory and drug addiction. The most scrutinized example of E-T coupling is Ca2+ signaling to the transcription factor CREB (cAMP response element-binding) protein via phosphorylation at Ser133. As an important source of Ca2+ influx, voltage-gated Ca2+ channels have been well studied for their biophysical and biochemical properties. Interestingly, in E-T coupling it seems that Ca2+ influxes through different Ca2+ channels can engage different signaling pathways to the nucleus. For example, CaV1 (also called L-type) channels enjoy a big advantage over CaV2 channels, even though CaV1 channels contribute only a minority of the overall Ca2+ entry in neurons. Our recent Cell paper uncovered that this disparity in potency can be explained by differences in how the two classes of Ca2+ channels employ local and global Ca2+ signaling. However, the 'private line' for the nanodomain advantage of CaV1 channels is unclear. Now we are poised to provide a detailed characterization of the critical question: what carries the long-distance signal from CaV1-anchored signaling complex to the nucleus? We have an answer: Ca2+/CaM translocation to the nucleus depends on a co-transporter that we now identify as γCaMKII. This shuttle gathers cytoplasmic Ca2+/CaM, sequestering it at the CaV1 channel before traveling to the nucleus under control of a nuclear localization signal. This signaling mechanism relies on γCaMKII, βCaMKII and CaN, signaling molecules that operate in the CaV1 nanodomain and also have been implicated in multiple neuropsychiatric diseases. This proposal focuses on understanding the cellular machinery of γCaMKII/CaM translocation and three specific aims are proposed. (1) Define the dynamics of Ca2+ signaling mechanisms that link CaV1 activity to nuclear CREB phosphorylation and CRE-dependent transcription. We will track γCaMKII translocation in real time and assess the impact of Ca2+/CaM delivered to the nucleus via this shuttle mechanism. (2) We will manipulate the γCaMKII pathway using genetic constructs in order to nail down the molecular components required for CREB phosphorylation. We will alter binding interactions and enzymatic actions involving CaM, βCaMKII, CaN, and PP2A at critical steps along the pathway. (3) Understand CaV1-dependent CaM shuttling in neocortical neurons and define distinct roles of nanodomain Ca2+ signaling and voltage gated conformational signaling for E-T coupling. Gaining a clearer picture of the linkage between CaV1 channels and CREB signaling will have a favorable impact on understanding how changes in gene expression alter the function of neurons in neural networks. Thus, the research is relevant both to basic cell biology and to disease states as diverse as addiction, autism and other neuropsychiatric diseases.