Phosphorylation-dependent regulation of calcium channels by macromolecular complexes

大分子复合物对钙通道的磷酸化依赖性调节

基本信息

批准号：
10161818
负责人：
Steven O Marx
金额：
$ 72.97万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10161818
关键词：
Action Potentials Address Adrenergic Agents Affect Alanine Arrhythmia Attenuated Binding Biochemical Bioinformatics Biotin Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Channel Cardiac Cardiac Myocytes Cardiovascular Diseases Catecholaminergic Polymorphic Ventricular Tachycardia Cavia Cell physiology Cells Complex Consensus Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic GMP Data Doxycycline Drug Targeting Electrophysiology (science)Exercise Failure Follow-Up Studies Goals Heart Heart Atrium Heart Diseases Heart failure Hormonal Hypertrophy Immunoprecipitation In Situ In Vitro Investigation Knock-out Knockout Mice Label Long QT Syndrome Macromolecular Complexes Maps Mass Spectrum Analysis Mediating Methods Modeling Molecular Molecular Probes Molecular Target Mus Muscle Cells N-terminal Oryctolagus cuniculus Pathology Pathway interactions Patients Permeability Peroxidases Phosphorylation Phosphorylation Site Physiological Porifera Process Proteins Proteome Regulation Ryanodine Receptors Serine Signal Pathway Site System Techniques Tertiary Protein Structure Testing Threonine Time Timothy syndrome Transgenic Mice Transgenic Organisms Troponin I ascorbate attenuation drug development fighting heart function in vivo inducible gene expression innovation insight knock-down mouse model mutant new therapeutic target novel phospholamban preservation prevent protein activation response small hairpin RNA small molecule tool

项目摘要

Our overall goal is to discover details of fundamental mechanisms underlying regulation of CaV1.2 channels that have eluded more than four decades of investigation. We propose to use novel tools and approaches to identify novel proteins, supramolecular complexes, and signaling pathways affecting CaV1.2 channels as the basis for targeted drug development for arrhythmias. Although it is well-established that phosphorylation by cyclic AMP (cAMP)-PKA, but not Ca2+/calmodulin kinase II (CaMKII), is the fundamental process by which b- adrenergic stimulation controls Ca2+ influx via CaV1.2 in the heart, the molecular targets of PKA remain unknown. A detailed molecular understanding of CaV1.2 regulation in myocytes has been hampered by the inability to recapitulate and then dissect in heterologous expression systems key aspects of CaV1.2 function in myocytes. Our novel tools surmount major obstacles that have limited progress in the field, and allow us to identify the neighboring proteome of CaV1.2 in the heart and probe molecular aspects of CaV1.2 regulation, using biochemical and electrophysiological techniques, within the context of cardiomyocytes, but with the power of a heterologous expression system. The failure thus far to identify any site as essential for adrenergic modulation led us to propose an alternative hypothesis: that a combination of phosphorylation sites in a1C is required for b-adrenergic stimulation of CaV1.2. To address this hypothesis, we generated mice with alanine- substitutions in rabbit a1C of all conserved and non-conserved consensus PKA phosphorylation sites (“35- mutant a1C”), and found that b-adrenergic regulation was not dependent upon any of these serine or threonine residues. Using a similar transgenic approach, we found that b-adrenergic regulation does not require phosphorylation of any of the 18 N-terminal, HOOK and GK domain consensus PKA phosphorylation sites in the b2b subunit. The next step is to create transgenic mice expressing b2b subunits with all PKA consensus sites removed (“33-mutant b2b”) and test regulation in a b2 knockout background. Thereafter, we will determine whether phosphorylation of either a1C or b subunits is sufficient to enable b-adrenergic regulation by crossing the 35-mutant a1C and the 33-mutant b2b mice. If adrenergic regulation is preserved, these results would shift a four-decade paradigm: the core CaV1.2 subunits are not the required PKA targets. Other aims of the proposal are to determine whether b-adrenergic stimulation of CaV1.2 is dependent upon a target extrinsic to CaV1.2 core subunits, and whether specifically attenuating b-adrenergic-modulation of CaV1.2 can suppress arrhythmogenesis. The feasibility of this approach is supported by the demonstration that disrupting the b-a interaction prevents b-adrenergic regulation of CaV1.2. The three Aims, which will provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding cardiac pathologies and the molecular mechanisms responsible for the modulation of cardiac contractility.

我们的总体目标是发现 CaV1.2 通道调控基本机制的细节我们建议使用新颖的工具和方法来解决这些问题。识别影响 CaV1.2 通道的新型蛋白质、超分子复合物和信号通路尽管磷酸化是心律失常靶向药物开发的基础。环 AMP (cAMP)-PKA，而不是 Ca2+/钙调蛋白激酶 II (CaMKII)，是 b- 的基本过程。肾上腺素能刺激通过心脏中的 CaV1.2 控制 Ca2+ 流入，PKA 的分子靶点仍然存在对肌细胞中 CaV1.2 调节的详细分子了解受到了阻碍。无法在异源表达系统中重述和剖析 CaV1.2 功能的关键方面我们的新工具克服了该领域进展有限的主要障碍，使我们能够识别心脏中 CaV1.2 的邻近蛋白质组并探索 CaV1.2 调节的分子方面，使用生化和电生理学技术，在心肌细胞的背景下，但与异源表达系统的力量迄今为止未能确定任何位点对于肾上腺素能至关重要。调节使我们提出了另一种假设：a1C 中磷酸化位点的组合是 CaV1.2 的 β 肾上腺素能刺激所需。为了解决这一假设，我们培育了具有丙氨酸的小鼠。兔 a1C 中所有保守和非保守共有 PKA 磷酸化位点的替换（“35- 突变体 a1C”），并发现 β-肾上腺素能调节不依赖于任何这些丝氨酸或苏氨酸使用类似的转基因方法，我们发现β-肾上腺素能调节不需要。 18 个 N 端、HOOK 和 GK 结构域共有 PKA 磷酸化位点中任意一个的磷酸化下一步是创建表达具有所有 PKA 共识的 b2b 亚基的转基因小鼠。删除位点（“33-突变体 b2b”）并在 b2 敲除背景中测试调节。此后，我们将确定。 a1C 或 b 亚基的磷酸化是否足以通过交叉作用实现 β 肾上腺素能调节 35 突变 a1C 和 33 突变 b2b 小鼠如果保留肾上腺素调节，这些结果将发生变化。四个十年范式：核心 CaV1.2 亚基不是所需的 PKA 目标该提案的其他目标。确定 CaV1.2 的 β-肾上腺素能刺激是否依赖于 CaV1.2 的外在靶标核心亚基，以及特异性减弱 CaV1.2 的 β-肾上腺素能调节是否可以抑制扰乱 b-a 的证明支持了这种方法的可行性。相互作用阻止 CaV1.2 的 β 肾上腺素调节，这将提供关键的新功能。关于心肌细胞 Ca2+ 内流调节的理解，与了解心脏病理学和负责调节心脏的分子机制收缩性。