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

描述（由申请人提供）：树突计算、活动依赖性基因表达、突触可塑性、学习和记忆是部分基于Ca2+通过CaV1.2电压门控l型Ca2+通道流入的神经元过程。CaV1.2通道的活性受其磷酸化状态的调节。脚手架蛋白AKAP79将调节激酶（蛋白激酶a， PKA）和磷酸酶（钙调磷酸酶，CaN）锚定在通道上。在海马锥体神经元中，去甲肾上腺素结合β肾上腺素能受体启动级联反应，导致cAMP的产生和PKA的激活。CaV1.2的磷酸化增强了通道活性，导致Ca2+内流增加。通道胞内口附近升高的Ca2+激活CaN磷酸酶，其作用是逆转通道磷酸化，从而下调通道活性并减少Ca2+内流。我们计划在显微镜下从单个活细胞中获得该信号复合体中不同伙伴之间荧光共振能量转移（FRET）的动态测量，以揭示通道调制基础的分子内和分子间运动。我们将及时将这些运动与CaV1.2电流的变化联系起来，我们将使用全细胞膜片钳记录同时测量CaV1.2电流。为了获得FRET测量，我们将荧光蛋白（青色CFP，黄色YFP）融合到各种信号伙伴（CaV1.2, AKAP79， PKA调节和催化亚基，CaN及其Ca2+依赖性激活剂钙调蛋白）。这些荧光融合构建体将被转染到HEK293细胞或培养的大鼠海马神经元中。我们将使用比率荧光法（CFP强度/YFP强度）来测量单个活细胞中信号伴侣之间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通道功能调制的机制。此外，分子复合体内的信号动力学，如本文提出的研究，有助于支持神经元中的高阶功能，如计算，因此这项工作的一般意义是广泛的。

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.