Calcium Channels, Calmodulin and Nuclear CREB Signaling

 描述（由申请人提供）：兴奋-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。这样，基因的表达可以以活性依赖性方式调节。神经元的重塑被认为是神经元发育、学习记忆和药物成瘾过程中长期适应性变化的必要和重要因素。E-T偶联的最详细的例子是Ca 2+通过Ser 133磷酸化向转录因子CREB（cAMP反应元件结合）蛋白质发出信号。电压门控性钙通道作为钙内流的重要来源，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Ca 2+通道流入的Ca 2+可以参与不同的信号通路到细胞核。例如，CaV 1（也称为L型）通道比CaV 2通道具有很大的优势，尽管CaV 1通道仅占神经元中总Ca 2+进入的一小部分。我们最近的Cell论文发现，这种效力的差异可以通过两类Ca 2+通道如何使用局部和全局Ca 2+信号的差异来解释。然而，CaV 1通道的纳米结构域优势的“专用线路”尚不清楚。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CaV 1锚定信号复合物传递到细胞核？我们有一个答案：Ca 2 +/CaM转运到细胞核取决于一种共转运蛋白，我们现在将其确定为γCaMKII。这种穿梭收集细胞质Ca 2 +/CaM，在细胞核定位信号的控制下，在前往细胞核之前将其隔离在CaV 1通道处。这种信号传导机制依赖于γCaMKII、βCaMKII和CaN，这些信号传导分子在CaV 1纳米结构域中起作用，也与多种神经精神疾病有关。该计划的重点是了解γCaMKII/CaM易位的细胞机制，并提出了三个具体目标。(1)定义将CaV 1活性与核CREB磷酸化和CREB依赖性转录联系起来的Ca 2+信号机制的动力学。我们将在真实的时间内跟踪γ CaMK II易位，并评估通过这种穿梭机制传递到细胞核的Ca 2 +/CaM的影响。(2)我们将使用基因构建体操纵γCaMKII通路，以确定CREB磷酸化所需的分子组分。我们将改变结合相互作用和酶作用，涉及钙调素，β CaMK Ⅱ，钙氮，和PP 2A在关键步骤沿着的途径。(3)了解CaV 1依赖性CaM在新皮层神经元中的穿梭，并定义纳米结构域Ca 2+信号传导和电压门控构象信号传导对E-T偶联的不同作用。更清楚地了解CaV 1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产生有利的影响。因此，该研究既与基本细胞有关， 生物学和疾病状态，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.