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: 10536570
• 负责人: Heather Spooner
• 金额: $ 3.91万
• 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/10536570
• 关键词: Address; Affect; Apoptosis; Basic Science; Behavior; Binding; Binding Sites; Biotinylation; C-terminal; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cells; Complex; Consensus; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Development; Electrophysiology (science); Forskolin; Goals; Heart; Heart Diseases; Investigation; Ion Channel; Ions; Isoproterenol; Knowledge; L-Type Calcium Channels; Link; Measures; Mediating; Microscopy; 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 Signaling; Regulation; Regulatory Pathway; Reporting; Resolution; 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; 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.

项目概述：电压门控L型钙通道（CaV1.2）对心脏兴奋至关重要- 该通道的收缩（EC）偶联和失调与许多形式的心脏病有关。14-3-3 是一种普遍存在的蛋白质，与许多细胞蛋白质相互作用，影响多种生理过程 包括细胞生长、凋亡和离子通道运输。优先结合磷酸丝氨酸/苏氨酸 靶蛋白上的残基以调节它们的运输、协同性、磷酸化状态和/或活性。在 HEK 293细胞，14-3-3增强另一种电压门控Ca 2+通道（CaV2.2）的运输，并已显示 通过与CaVβ亚基的相互作用间接改变CaV 1.2的运输，然而，直接证据和信息 关于这种调节的程度和磷酸化依赖性仍然是需要的。此外， 没有研究14-3-3在心肌细胞中CaV1.2通道运输/调节中的作用。我们解决 这些知识的差距在当前的应用。自14-3-3以来，已报告，以促进合作 门控的电压依赖性心脏Na+通道，NaV1.5，我们还将研究14-3-3在 CaV1.2的协同作用。当变构相互作用形成时，CaV1.2的这种门控模式发生 在簇中的相邻通道的C-末端尾部之间，使得一个通道的开口可以 与其他附着通道连通，以提高其开放概率并放大全细胞Ca 2+内流。 我们的小组先前已经表明，PKA介导的CaV1.2通道磷酸化触发增强的CaV1.2通道磷酸化。 将这些通道运输到心室肌细胞的肌膜中，产生更大的通道簇， 促进增强的协同门控行为和增强的全细胞Ca 2+电流。这有助于调整心脏 EC耦合，以满足战斗或飞行期间的增强需求。然而，这种增强的分子细节 贩运不明确。在这里，我们提出14-3-3在这种反应中起作用。我们发现了几个 CaV1.2的C-尾上14-3-3的推定结合位点和其他关键调控位点，包括共有位点 PKA磷酸化位点。该项目旨在测试14-3-3调节CaV1.2运输的假设， 导致肌膜上的增强的通道聚集，这促进了协作性相互作用， 增加Ca 2+内流。我们进一步提出，这些相互作用是由通道加强 磷酸化提供了调节CaV1.2通道活性和EC偶联以满足需求的手段。在 在这个为期两年的博士前项目中，我们将在三个具体目标中严格检验这一假设。目标1测试 假设14-3-3以磷酸化依赖性方式与CaV1.2相互作用。目标2检验假设 CaV1.2通道运输、肌膜聚集和合作相互作用被14-3-3增强。 目的3关注这种调节对心脏EC偶联的功能影响。CaV1.2通道改变 运输和调节与许多心肌病有关，因为它在心脏EC中起着核心作用， 因此，我们的目标与NHLBI的使命相关。