Investigating Cardiac Ion Channels by Novel Methods

通过新方法研究心脏离子通道

基本信息

批准号：
10418713
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
项目类别：
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/10418713
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 mutant new therapeutic target novel prevent protein activation reconstitution recruit response voltage

项目摘要

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调节的生理稳态机制 CaV1.2 在心脏中的通道并确定心力衰竭和心律失常的新治疗靶点。 L型Ca2+通道在心脏兴奋-收缩耦合中起关键作用，是心脏兴奋和收缩耦合的重要靶点。交感神经系统和一些信号通路在“战斗或-”过程中增加心肌收缩力。飞行反应是由转基因小鼠心脏中 CaV1.2 通道的 β-肾上腺素增强引起的。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，最近，我们确定了 β-肾上腺素能的机制。激动剂刺激电压门控 Ca2+ 通道，我们表达与抗坏血酸缀合的 α1C 或 β2B 亚基。小鼠心脏中的过氧化物酶，并使用多重定量蛋白质组学来追踪小鼠心脏中的数百种蛋白质我们观察到 Ca2+ 通道抑制剂 Rad（一种单体 G 蛋白）与 CaV1.2 非常接近。在 CaV1.2 微环境中富集，但在 PKA 催化过程中耗尽。 Rad 上特定 Ser 残基的磷酸化会降低其对辅助 β 亚基的亲和力并缓解 CaV1.2 的组成型抑制表现为通道开放概率的增加。我们提出了三个目标： (1)使用Rad的4个PKA磷酸化位点突变为丙氨酸的敲入小鼠，以及不能结合 Rad 的突变 CaVβ 亚基的心脏特异性表达，我们将在 (2)心肌细胞具有Rad磷酸化在体内调节心肌收缩力的作用。成功应用邻近标记，我们现在还建议鉴定 A-激酶锚定蛋白 (AKAP) 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-肾上腺素能调节尚不清楚 (3) cGMP 激活 PKG。抑制 CaV1.2 并抵消心肌细胞中 Ca2+ 电流的 β-肾上腺素刺激。因此，激活可以作为 CaV1.2 肾上腺素刺激的靶向抑制剂以及伴随的我们勇敢地承认 PKG 信号传导至少可以阻断 β-肾上腺素能诱导的 CaV1.2 刺激。多种机制之一：i) 通过 α1C 或 β2B 的直接 PKG 磷酸化；ii) 通过阻止 α1C 或 β2B 的募集； PKA 作用于 CaV1.2 复合物；iii) 通过阻止 Rad 在心脏中与 CaV1.2 复合物解离。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体 α1C 和具有正常 β-肾上腺素能刺激 CaV1.2 的 β2B 转基因小鼠剖析上游信号传导。途径，我们将利用邻近蛋白质组学的三个目标，这将提供关键的新理解。关于心肌细胞中 Ca2+ 内流的调节，与理解心肌细胞高度相关负责调节心肌收缩力和心律失常发生的分子机制。