Reciprocal Control of Ca2+ Channels by Anchored Protein Kinase A and Calcineurin

描述(申请人提供)：树突计算、活性依赖的基因表达、突触可塑性、学习和记忆是部分基于钙离子通过CaV1.2电压门控L型钙离子通道内流的神经元过程。CaV1.2通道的活性受其磷酸化状态的调节。支架蛋白AKAP79将一个调节激酶(蛋白激酶A，PKA)和一个磷酸酶(钙调神经磷酸酶，CaN)锚定在通道上。在海马区锥体神经元中，去甲肾上腺素与β-肾上腺素能受体结合后启动一系列的信号传导，导致cAMP的产生和PKA的激活。CaV1.2的磷酸化增强了通道的活性，导致钙内流增加。靠近通道胞内口的高钙激活了CaN磷酸酶，从而逆转了通道的磷酸化，从而下调了通道的活性，减少了钙的内流。我们计划在显微镜舞台上从单个活细胞上获得这个信号复合体中不同伙伴之间的荧光共振能量转移(FRET)的动态测量，以努力揭示通道调制背后的分子内和分子间运动。我们将在时间上将这些运动与CaV1.2电流的变化相关联，我们将使用全细胞膜片钳记录同时测量这些变化。为了获得FRET测量，我们将荧光蛋白(青色CFP，黄色YFP)与不同的信号伙伴(CaV1.2，AKAP79，PKA调节和催化亚基，CaN及其钙依赖的激活剂钙调蛋白)融合。这些荧光融合构建体将被导入HEK293细胞或培养的大鼠海马神经元。我们将使用比率荧光方法(CFP强度/YFP强度)来测量单个活细胞中信号伙伴之间FRET的动态变化。该研究计划的目标是在分子水平上深入了解CaV1.2通道功能的调节机制。此外，分子复合体中信号的动力学，如这里提出的研究，有助于支持神经元的高阶功能，如计算，因此这项工作的一般意义是广泛的。 与公共卫生的相关性。钙通道有助于控制神经细胞的活动，心脏细胞的跳动，包裹在血管周围的平滑肌细胞的收缩，以及胰腺胰岛素的分泌。因此，钙通道是药物的靶点，用于预防中风后神经细胞死亡，治疗心律失常和心绞痛，以及对抗高血压。这里研究的钙通道突变已被确定为人类Timothy综合征的原因，其症状是认知障碍和潜在的致命的心律失常。

DESCRIPTION (provided by applicant): Dendritic computation, activity-dependent gene expression, synaptic plasticity, learning and memory are neuronal processes that are based in part upon Ca2+ influx through CaV1.2 voltage-gated L-type Ca2+ channels. The activity of CaV1.2 channels is modulated by their phosphorylation state. The scaffolding protein AKAP79 anchors both a regulating kinase (protein kinase A, PKA) and a phosphatase (calcineurin, CaN) to the channel. In hippocampal pyramidal neurons, norepinephrine binding to beta adrenergic receptors initiates a cascade that leads to cAMP production and PKA activation. Phosphorylation of CaV1.2 enhances channel activity, resulting in increased Ca2+ influx. The elevated Ca2+ near the intracellular mouth of the channel activates the CaN phosphatase, which acts to reverse channel phosphorylation and consequently down-modulate channel activity and reduce Ca2+ influx. We plan to obtain from single living cells on a microscope stage dynamic measurements of fluorescence resonance energy transfer (FRET) between the various partners in this signaling complex, in an effort to uncover intra- and intermolecular movements that underlie channel modulation. We will correlate in time these movements with changes in CaV1.2 current that we will simultaneously measure using whole-cell patch-clamp recording. To obtain FRET measurements, we will fuse fluorescent proteins (cyan CFP, yellow YFP) to various pairs of signaling partners (CaV1.2, AKAP79, PKA regulatory and catalytic subunits, CaN and its Ca2+-dependent activator calmodulin). These fluorescent fusion constructs will be transfected into HEK293 cells or cultured rat hippocampal neurons. We will use a ratiometric fluorescence approach (CFP intensity/YFP intensity) to measure dynamic changes in FRET between signaling partners in single live cells. The goal of the research plan is to gain substantial insight into the mechanics, at the molecular level, of modulation of CaV1.2 channel function. In addition, the dynamics of signaling within molecular complexes, such as the one proposed for study here, helps support higher order function in neurons, such as computation, and thus the general significance of the work is broad. Relevance to Public Health. Calcium channels help control activity of nerve cells, beating of heart cells, contraction of smooth muscle cells that are wrapped around blood vessels, and secretion of insulin from the pancreas. Calcium channels are therefore targets of drugs used to protect against nerve cell death following stroke, to treat cardiac arrhythmias and angina, and to combat high blood pressure. A mutation in the calcium channel studied here has been identified as the cause of Timothy syndrome in humans, symptoms of which are cognitive impairment and potentially fatal disturbance of heart rhythm.