Elucidating the mechanism of beta-adrenergic regulation in L-type Calcium Channels (CaV1.2)

阐明 L 型钙通道 (CaV1.2) 中 β-肾上腺素能调节的机制

• 批准号: 10490970
• 负责人: Arianne Papa
• 金额: $ 0.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490970
• 关键词: Adrenergic Agents; Adrenergic beta-Agonists; Alanine; Arrhythmia; Attenuated; Ca(2+)-Transporting ATPase; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Cells; Characteristics; Cyclic AMP-Dependent Protein Kinases; Data; Electrophysiology (science); Enzymes; Exercise; Exons; Forskolin; Goals; Heart; Heart Atrium; Heart Hypertrophy; Heart failure; In Vitro; Investigation; Knock-in; Knock-in Mouse; L-Type Calcium Channels; Label; Laboratories; Lead; Link; Macromolecular Complexes; Mass Spectrum Analysis; Measurement; Mediating; Methods; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Pacemakers; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiology; Play; Proteomics; Regulation; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sinus; Surface; Sympathetic Nervous System; System; Techniques; Testing; base; calmodulin-dependent protein kinase II; design; experience; experimental study; fighting; heart function; hormone regulation; in vivo; inhibitor; mouse model; mutant; novel; novel strategies; phospholamban; prevent; protein activation; reconstitution; response; skills; trafficking; voltage; Adrenergic Agents; Adrenergic beta-Agonists; Alanine; Arrhythmia; Attenuated; Ca(2+)-Transporting ATPase; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Cells; Characteristics; Cyclic AMP-Dependent Protein Kinases; Data; Electrophysiology (science); Enzymes; Exercise; Exons; Forskolin; Goals; Heart; Heart Atrium; Heart Hypertrophy; Heart failure; In Vitro; Investigation; Knock-in; Knock-in Mouse; L-Type Calcium Channels; Label; Laboratories; Lead; Link; Macromolecular Complexes; Mass Spectrum Analysis; Measurement; Mediating; Methods; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Pacemakers; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiology; Play; Proteomics; Regulation; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sinus; Surface; Sympathetic Nervous System; System; Techniques; Testing; base; calmodulin-dependent protein kinase II; design; experience; experimental study; fighting; heart function; hormone regulation; in vivo; inhibitor; mouse model; mutant; novel; novel strategies; phospholamban; prevent; protein activation; reconstitution; response; skills; trafficking; voltage

Project Summary My overall goal is to uncover mechanisms responsible for physiological regulation of the voltage-gated calcium channel in the heart. Calcium influx through voltage-gated L-type calcium channels (CaV1.2) is an essential signal initiating each heartbeat. Dysfunctional calcium channel trafficking and regulation have been implicated in the mechanisms of arrhythmias, cardiac hypertrophy, and heart failure. During the “fight or flight” response, beta-adrenergic activation of protein kinase A (PKA) increases this calcium influx and increases cardiac contractility. Despite decades of investigation, the detailed mechanism by which this pathway activates calcium channels in the heart remains unknown. Strong preliminary data in the laboratory suggest that the calcium channel inhibitor Rad, a small G-protein, is the missing link that enables PKA regulation of CaV1.2. Based on proximity proteomics, Rad is enriched in the CaV1.2 microenvironment but is depleted during beta- adrenergic stimulation in the heart. We confirmed in a heterologous expression system that Rad co-expression fully-reconstituted PKA modulation at the whole-cell level and recapitulated single-channel characteristics of PKA modulation. Furthermore, we demonstrated that Rad is also the key functional target of PKA phosphorylation, as eliminating Rad phosphorylation sites abolished forskolin-mediated stimulation of the calcium channels. In the end, the underlying mechanism turns out to be simple and elegant – Rad at baseline inhibits CaV1.2 activity, while PKA phosphorylation of Rad relieves this inhibition. My hypothesis is that Rad phosphorylation is sufficient for calcium channel regulation in the heart, and that loss of beta-adrenergic regulation of calcium channels attenuates adrenergic-induced increase in inotropy. essential component of beta-adrenergic regulation, Via two Aims, I will: (1) validate Rad phosphorylation as an and (2) assess the contribution of PKA-induced stimulation of calcium currents in forming the cardiac response to beta-adrenergic agonists. Both Aims utilize novel knock- in mice, and require cellular electrophysiological techniques and in vivo measurements of cardiac function. The two Aims will identify new mechanisms responsible for regulation of calcium influx in cardiomyocytes, which may lead to novel approaches to modulate cardiac contractility and arrhythmias.

项目摘要 我的总体目标是发现负责电压门控物理调节的机制 心脏中的钙通道。通过电压门控L型钙通道（CAV1.2）的钙影响是一种 基本信号启动每个心跳。功能失调的钙通道运输和调节 以心律不齐，心脏肥大和心力衰竭的机制实施。在“战斗或飞行”期间 反应，蛋白激酶A（PKA）的β-肾上腺素能激活增加了钙的影响并增加 心脏收缩。尽管进行了数十年的调查，但该途径激活的详细机制 心脏中的钙通道仍然未知。实验室中强大的初步数据表明 钙通道抑制剂RAD是一种小的G蛋白，是毫无疑问的链路，可用于PKA调节CAV1.2。 基于接近度蛋白质组学，RAD富含CAV1.2微环境，但在β- 心脏中的肾上腺素刺激。我们在异源表达系统中确认RAD共表达 在整个细胞水平上的完全稳定的PKA调制和PKA的概括单通道特性 调制。此外，我们证明RAD也是PKA磷酸化的关键功能靶标，即 由于消除了RAD磷酸化位点，消除了钙通道的福尔司蛋白介导的刺激。在 末端，基本机制很简单，优雅 - 基线时RAD抑制Cav1.2活性， 而RAD的PKA磷酸化挽救了这种抑制作用。我的假设是RAD磷酸化足够 对于心脏中的钙通道调节，钙通道的β-肾上腺素能调节的丧失 减弱肾上腺素引起的肌力增加。 β-肾上腺素能调节的重要组成部分， 通过两个目的，我将：（1）验证RAD磷酸化为一个 （2）评估PKA诱导的模拟的贡献 钙电流形成对β-肾上腺素激动剂的心脏反应。两者的目标都利用新颖的敲门 在小鼠中，需要细胞电生理技术和心脏功能的体内测量。 两个目标将确定负责调节心肌细胞钙影响的新机制，这可能 导致新的方法调节心脏收缩和心律不齐。