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

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.

项目概要 我的总体目标是揭示负责电压门控生理调节的机制 心脏中的钙通道通过电压门控 L 型钙通道 (CaV1.2) 流入。 启动每次心跳的重要信号钙通道运输和调节功能失调。 参与“战斗或逃跑”期间的心律失常、心脏肥大和心力衰竭的机制。 响应，蛋白激酶 A (PKA) 的 β-肾上腺素能激活会增加钙流入并增加 尽管经过数十年的研究，该途径激活的详细机制。 实验室中强有力的初步数据表明，心脏中的钙通道仍然未知。 钙通道抑制剂 Rad 是一种小 G 蛋白，是 PKA 调节 CaV1.2 的缺失环节。 基于邻近蛋白质组学，Rad 在 CaV1.2 微环境中富集，但在 beta- 我们在异源表达系统中证实了 Rad 的共表达。 全细胞水平上完全重构的 PKA 调制和概括的 PKA 单通道特征 此外，我们证明 Rad 也是 PKA 磷酸化的关键功能靶点， 因为消除 Rad 磷酸化位点就消除了毛喉素介导的钙通道刺激。 最后，底层机制被证明是简单而优雅的——基线时的 Rad 抑制 CaV1.2 活性， 而 Rad 的 PKA 磷酸化可以缓解这种抑制。我的假设是 Rad 磷酸化就足够了。 心脏钙通道调节，以及钙通道β-肾上腺素调节的丧失 减弱肾上腺素能引起的正性肌力增加。 β-肾上腺素能调节的重要组成部分， 通过两个目标，我将：(1) 验证 Rad 磷酸化作为 (2) 评估 PKA 诱导刺激的贡献 钙电流在形成对β-肾上腺素能激动剂的心脏反应中的作用。这两个目标都利用了新颖的敲击- 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流入的新机制，这可能 导致调节心肌收缩力和心律失常的新方法。