14-3-3 regulation of cardiac L-type calcium channels and EC-coupling

14-3-3 心脏 L 型钙通道和 EC 偶联的调节

• 批准号: 10753500
• 负责人: Heather Spooner
• 金额: $ 4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10753500
• 关键词: Address; Affect; Apoptosis; Basic Science; Behavior; Binding; Binding Sites; Biotinylation; C-terminal; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Co-Immunoprecipitations; Communication; Complex; Consensus; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Development; Electrophysiology (science); Endosomes; Forskolin; Goals; Heart; Heart Diseases; Investigation; Ion Channel; Isoproterenol; Knowledge; L-Type Calcium Channels; Link; Measures; Mediating; Mission; Modality; Modeling; Molecular; Muscle Cells; National Heart, Lung, and Blood Institute; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphoserine; Physiological Processes; Play; Probability; Property; Protein Isoforms; Proteins; Receptor Activation; Receptor Signaling; Regulation; Regulatory Pathway; Reporting; Role; Sarcolemma; Signal Pathway; Site; Sodium Channel; Surface; Tail; Testing; Threonine; Ventricular; adenoviral mediated; beta-adrenergic receptor; cell growth; density; fighting; hemodynamics; insight; nanoscale; new therapeutic target; overexpression; pre-doctoral; protein function; response; superresolution microscopy; trafficking; voltage

Project Summary: The voltage-gated L-type calcium channel (CaV1.2) is essential for cardiac excitation- contraction (EC)-coupling and dysregulation of the channel is implicated in many forms of heart disease. 14-3-3 is a ubiquitous protein that interacts with numerous cellular proteins to affect multiple physiological processes including cell growth, apoptosis, and ion channel trafficking. It preferentially binds phospho-serine/threonine residues on target proteins to regulate their trafficking, cooperativity, phosphorylation state, and/or activity. In HEK293 cells, 14-3-3 enhances trafficking of another voltage gated Ca2+ channel (CaV2.2) and has been shown to indirectly alter CaV1.2 trafficking via interactions with CaVβ subunits, however direct evidence and information about the extent and phosphorylation-dependence of this regulation is still needed. In addition, there have been no investigations into the role of 14-3-3 in CaV1.2 channel trafficking/regulation in cardiomyocytes. We address these gaps in knowledge in the current application. Since 14-3-3 has been reported to facilitate cooperative gating of the voltage-dependent cardiac Na+ channel, NaV1.5, we will also investigate the role of 14-3-3 in cooperative interactions of CaV1.2. This gating modality of CaV1.2 occurs when allosteric interactions form between C-terminal tails of adjacent channels in a cluster such that the opening of one channel can be communicated to other attached channels to enhance their open probability and amplify whole-cell Ca2+ influx. Our group has previously shown that PKA-mediated phosphorylation of CaV1.2 channels triggers enhanced trafficking of these channels into the sarcolemma of ventricular myocytes, producing larger channel clusters that facilitate enhanced cooperative gating behavior and augmented whole-cell Ca2+ currents. This helps tune cardiac EC-coupling to meet the enhanced demand during fight-or-flight. However, the molecular details of this enhanced trafficking are unclear. Here we propose that 14-3-3 plays a role in this response. We have identified several putative binding sites for 14-3-3 on the C-tail of CaV1.2 and other critical regulatory sites, including consensus PKA phosphorylation sites. This project aims to test the hypothesis that 14-3-3 regulates CaV1.2 trafficking, resulting in enhanced channel clustering on the sarcolemma that facilitates cooperative interactions and amplifies Ca2+ influx. We further propose that these interactions are strengthened by channel phosphorylation providing a means to tune CaV1.2 channel activity and EC-coupling to meet demand. In this two-year predoctoral project, we will rigorously test this hypothesis in three Specific Aims. Aim 1 tests the hypothesis that 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner. Aim 2 tests the hypothesis that CaV1.2 channel trafficking, sarcolemmal clustering, and cooperative interactions are enhanced by 14-3-3. Aim 3 focuses on the functional effects of this regulation on cardiac EC-coupling. Alterations in CaV1.2 channel trafficking and regulation are associated with numerous cardiomyopathies given its central role in cardiac EC- coupling; thus our goals are relevant to the mission of the NHLBI.

项目摘要：电压门控L型钙通道（CAV1.2）对于心脏兴奋至关重要 - 通道的收缩（EC） - 偶联和失调以多种形式的心脏病实施。 14-3-3 是一种无处不在的蛋白质，与许多细胞蛋白相互作用以影响多种物理过程 包括细胞生长，凋亡和离子通道运输。它优先结合磷酸丝氨酸/苏氨酸 靶蛋白上的残基调节其运输，协调，磷酸化状态和/或活动。在 HEK293细胞，14-3-3增强了另一个电压门控Ca2+通道（CAV2.2）的运输，已显示 通过与CAVβ亚基相互作用间接改变CAV1.2运输，但是直接证据和信息 仍然需要该调节的程度和磷酸化依赖性。此外，还有 在CAV1.2频道贩运/监管心肌细胞中，没有对14-3-3的作用进行投资。我们解决 当前应用程序中的知识中的这些差距。自从据报道以来14-3-3以来可以促进合作 电压依赖性心脏Na+通道的门控NAV1.5，我们还将研究14-3-3的作用 Cav1.2的合作互动。当变构相互作用形成时，CAV1.2的门控方式发生 在一个集群中相邻通道的C末端尾巴之间，使一个通道的打开可以是 通信到其他附加的通道，以增强其开放概率并扩大全细胞Ca2+影响。 我们的小组先前已经表明，PKA介导的CAV1.2通道的磷酸化触发了增强 将这些通道贩运到心室心肌细胞的肌肉中，产生较大的通道簇 促进增强的教练行为并增强全细胞Ca2+电流。这有助于调整心脏 EC耦合满足战斗或飞行期间的增强需求。但是，此增强的分子细节 贩运尚不清楚。在这里，我们建议14-3-3在这一反应中起作用。我们已经确定了几个 在CAV1.2和其他关键调节位置的C尾上，定义的结合位点，包括共识 PKA磷酸化位点。该项目旨在检验14-3-3调节Cav1.2贩运的假设， 导致在肌膜上增强的渠道聚类，从而有助于合作互动和 放大器CA2+影响。我们进一步建议，这些相互作用得到了渠道的加强 磷酸化提供了一种调整CAV1.2通道活性和EC偶联以满足需求的方法。 这个为期两年的专业项目，我们将以三个特定的目的严格检验这一假设。 AIM 1测试 假设14-3-3以磷酸化依赖性方式与CAV1.2相互作用。 AIM 2检验假设 14-3-3增强了CAV1.2渠道贩运，肌肉聚类和合作相互作用。 AIM 3重点介绍了该调节对心脏EC偶联的功能效应。 CAV1.2通道的改变 由于其在心脏EC-中的核心作用 耦合;因此，我们的目标与NHLBI的使命有关。