Investigating Cardiac Ion Channels by Novel Methods

通过新方法研究心脏离子通道

• 批准号: 10219521
• 负责人: Steven O Marx
• 金额: $ 58.75万
• 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: 2021-06-05 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10219521
• 关键词: A kinase anchoring protein; Address; Adrenergic Agents; Adrenergic beta-Agonists; Affinity; Agonist; Alanine; Arrhythmia; Binding; Biochemical; Biotin; Cardiac; Cardiac Myocytes; Cells; Characteristics; Complex; Coupling; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dissociation; Doxycycline; Electron Transport Complex III; Electrophysiology (science); Enzymes; Exercise; Forskolin; Goals; Heart; Heart failure; Ion Channel; Knock-in Mouse; Knockout Mice; Label; Macromolecular Complexes; Mass Spectrum Analysis; Measurement; Mediating; Methodology; Methods; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Mutate; Neighborhoods; Peptides; Peroxidases; Phosphorylation; Phosphorylation Site; Physiological; Play; Probability; Process; Proteins; Proteomics; Regulation; Resistance; Role; Sarcoplasmic Reticulum; Serine; Signal Pathway; Signal Transduction; Sympathetic Nervous System; Transgenic Mice; Transgenic Organisms; ascorbate; experience; fighting; in vivo; inhibitor/antagonist; mutant; new therapeutic target; novel; prevent; protein activation; reconstitution; recruit; response; voltage

Our long-term overall goals are to discover physiologic homeostatic mechanisms underlying regulation of CaV1.2 channels in the heart and to identify novel therapeutic targets for heart failure and arrhythmias. CaV1.2, the L-type Ca2+ channel that plays a key role in cardiac excitation-contraction coupling, is an important target of the sympathetic nervous system and several signaling pathways. Increased cardiac contractility during fight-or- flight response is caused by β-adrenergic augmentation of CaV1.2 channels. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant CaV1.2 α1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Recently, we identified the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed α1C or β2B subunits conjugated to ascorbate- peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in close proximity to CaV1.2. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-subunits and relieves constitutive inhibition of CaV1.2 observed as an increase in channel open probability. We propose three Aims: (1) Using knock-in mice with the four PKA phosphorylation sites of Rad mutated to alanine, and mice with cardiac-specific expression of a mutant CaVβ subunit that cannot bind Rad, we will determine in cardiomyocytes the role of Rad phosphorylation in regulating cardiac contractility in vivo. (2) Having successfully applied proximity labeling, we now also propose to identify the A-kinase anchoring proteins (AKAPs) that facilitate β-adrenergic regulation of CaV1.2 in cardiomyocytes. The identity of the AKAP that facilitates β-adrenergic regulation of CaV1.2 in cardiomyocytes is unknown. (3) PKG activation by cGMP inhibits CaV1.2 and counteracts β-adrenergic stimulation of Ca2+ current in cardiomyocytes. Strategic PKG activation could therefore serve as a targeted suppressor of adrenergic stimulation of CaV1.2 and concomitant arrhythmias. We hypothesize that PKG signaling blocks β-adrenergic-induced stimulation of CaV1.2 by at least one of several mechanisms: i) by direct PKG phosphorylation of α1C or β2B; ii) by preventing the recruitment of PKA to the CaV1.2 complex; iii) by preventing the dissociation of Rad from the CaV1.2 complex in the heart. To assess whether PKG phosphorylation of α1C or β2B is required, we will utilize our fully phospho-mutant α1C and β2B transgenic mice that have normal β-adrenergic stimulation of CaV1.2. To dissect the upstream signaling pathways, we will utilize proximity proteomics. The three Aims, which will provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding the molecular mechanisms responsible for the modulation of cardiac contractility and arrhythmogenesis.

我们的长期总体目标是发现生理稳态机制的调节， CaV1.2通道的心脏，并确定新的治疗目标，心力衰竭和心律失常。CaV1.2， L-型钙通道在心脏兴奋-收缩偶联中起关键作用，是心肌缺血的重要靶点。 交感神经系统和几个信号通路。在战斗或- 飞行反应是由CaV1.2通道的β-肾上腺素能增强引起的。在转基因小鼠心脏中 完全表达PKA磷酸化位点缺陷突变体CaV1.2 α1C和β亚基，这种调节持续存在， 这意味着涉及额外的渠道因素。最近，我们确定了β-肾上腺素能神经元 激动剂刺激电压门控Ca 2+通道。我们表达了与抗坏血酸结合的α1C或β2B亚基， 过氧化物酶在小鼠心脏和使用多重，定量蛋白质组学跟踪数百种蛋白质， 接近CaV1.2。我们观察到钙通道抑制剂Rad，一种单体G蛋白， 在CaV1.2微环境中富集，但在β-肾上腺素能刺激期间耗尽。PKA催化 Rad上特异性Ser残基的磷酸化降低了其对辅助β亚基的亲和力， CaV1.2的组成性抑制被观察为通道开放概率的增加。我们提出三个目标： (1)使用Rad的四个PKA磷酸化位点突变为丙氨酸的基因敲入小鼠和 心脏特异性表达不能结合Rad的突变CaVβ亚基，我们将确定在 在体内研究了Rad磷酸化在调节心肌收缩力中的作用。(2)具有 成功地应用邻近标记，我们现在也提出了确定A-激酶锚定蛋白 （AKAP），其促进心肌细胞中CaV1.2的β-肾上腺素能调节。AKAP的身份， 促进心肌细胞中CaV1.2的β-肾上腺素能调节。(3)cGMP激活PKG 抑制CaV1.2和抵消心肌细胞中β-肾上腺素能刺激的Ca 2+电流。战略包装 因此，激活可以作为CaV1.2的肾上腺素能刺激的靶向抑制剂， 心律不齐我们假设PKG信号至少通过以下途径阻断β-肾上腺素能诱导的CaV1.2刺激： 几种机制之一：i）通过α1C或β2B的直接PKG磷酸化; ii）通过阻止 PKA与CaV1.2复合物的结合; iii）通过防止Rad在心脏中从CaV1.2复合物解离。到 评估是否需要α1C或β2B的PKG磷酸化，我们将利用我们的完全磷酸化突变的α1C， 具有正常CaV1.2的β-肾上腺素能刺激的β2B转基因小鼠。分析上行信号 我们将利用邻近蛋白质组学。三个目标，这将提供关键的新的理解 关于心肌细胞中Ca 2+内流的调节，与理解心肌细胞中Ca 2+内流的机制高度相关。 负责调节心脏收缩力和血管生成的分子机制。