Investigating Cardiac Ion Channels by Novel Methods

通过新方法研究心脏离子通道

• 批准号: 10673191
• 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/10673191
• 关键词: A kinase anchoring protein; Ablation; 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; 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; mutant; new therapeutic target; novel; prevent; protein activation; protein 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对生理稳态机制的规定 CAV1.2心脏中的通道并确定心力衰竭和心律不齐的新型治疗靶标。 Cav1.2， L型Ca2+通道在心脏兴奋 - 收缩耦合中起着关键作用，是 交感神经系统和几种信号通路。在战斗中提高心脏收缩性 飞行反应是由Cav1.2通道的β-肾上腺素能增强引起的。在转基因鼠心 表达完全PKA磷酸化位点缺陷的突变体CAV1.2α1C和β亚基，该调节持续存在， 意味着涉及大道外因素。最近，我们确定了β-肾上腺素的机制 激动剂刺激电压门控Ca2+通道。我们表达了与抗坏血酸 - 小鼠心脏中的过氧化物酶，并使用多路复用的定量蛋白质组学跟踪数百种蛋白质 靠近Cav1.2。我们观察到Ca2+通道抑制剂RAD是一种单体G蛋白，是 富含CAV1.2微环境，但在β-肾上腺素能刺激期间耗尽。 PKA催化 在RAD上的特定Ser保留的磷酸化降低了其对辅助β-亚基和救援的亲和力 构成对CAV1.2的抑制是通道开放概率增加的。我们提出了三个目标： （1）使用RAD突变为丙氨酸的RAD的四个PKA磷酸化位点，使用敲入小鼠，而小鼠则 突变体CAVβ亚基的心脏特异性表达无法结合RAD，我们将在 心肌细胞RAD磷酸化在体内调节心脏收缩率中的作用。 （2）有 成功应用接近标记，我们现在还建议识别锚定蛋白的A-激酶 （AKAP）促进心肌细胞中CAV1.2的β-肾上腺素能调节。 AKAP的身份 尚不清楚心肌细胞中CAV1.2的β-肾上腺素能调节。 （3）CGMP激活PKG 抑制CAV1.2并抵消心肌细胞中Ca2+电流的β-肾上腺素能刺激。战略性PKG 因此，激活可以用作CAV1.2肾上腺刺激的靶向抑制和伴随的抑制 心律不齐。我们假设PKG信号传导至少会阻止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）防止招募 PKA至CAV1.2复合物； iii）通过防止RAD从心脏中的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突变剂α1C和 β2b转基因小鼠的cav1.2具有正常的β-肾上腺素能刺激。剖析上游信号 途径，我们将利用接近蛋白质组学。这三个目标将提供关键的新理解 关注心肌细胞中Ca2+影响的调节，与理解 负责调节心脏收缩和心律失常发生的分子机制。