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) 耦合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，可与多种细胞蛋白质相互作用，影响多种生理过程 包括细胞生长、细胞凋亡和离子通道运输。它优先结合磷酸丝氨酸/苏氨酸。 靶蛋白上的残基以调节其运输、合作、磷酸化状态和/或活性。 HEK293 细胞 14-3-3 增强了另一个电压门控 Ca2+ 通道 (CaV2.2) 的运输，并且已被证明 通过与 CaVβ 亚基相互作用间接改变 CaV1.2 的运输，但是直接证据和信息 此外，还需要了解这种调节的程度和磷酸化依赖性。 尚未研究 14-3-3 在心肌细胞 CaV1.2 通道运输/调节中的作用。 自 14-3-3 以来，这些知识差距已被报告以促进合作。 电压依赖性心脏 Na+ 通道 NaV1.5 的门控，我们还将研究 14-3-3 在 CaV1.2 的这种门控模式在变构相互作用形成时发生。 簇中相邻通道的 C 端尾部之间，使得一个通道的开口可以是 与其他附着通道通讯，以提高其开放概率并放大全细胞 Ca2+ 流入。 我们的小组之前已经表明，PKA 介导的 CaV1.2 通道磷酸化触发增强 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 Ca2+ 电流，这有助于调节心脏。 EC耦合可以满足战斗或逃跑过程中增强的需求，但是，这种增强的分子细节。 在此我们提出 14-3-3 在这一应对中发挥了作用，我们已经确定了几个。 CaV1.2 C 尾上 14-3-3 的推定结合位点和其他关键调控位点，包括共有序列 该项目旨在检验 14-3-3 调节 CaV1.2 运输的假设。 导致肌膜上的通道聚集增强，促进合作互动和 我们进一步提出这些相互作用通过通道得到加强。 磷酸化提供了一种调节 CaV1.2 通道活性和 EC 偶联以满足 In 需求的方法。 在这个为期两年的博士前项目中，我们将在三个具体目标 1 测试中严格检验这一假设。 假设 14-3-3 以磷酸化依赖性方式与 CaV1.2 相互作用，目标 2 检验该假设。 14-3-3 增强了 CaV1.2 通道运输、肌膜簇和合作相互作用。 目标 3 重点关注这种调节对 CaV1.2 通道心脏 EC 耦合变化的功能影响。 鉴于其在心脏 EC-中的核心作用，转运和调节与许多心肌病相关。 耦合；因此我们的目标与 NHLBI 的使命相关。