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

项目摘要：电压门控L型钙通道(CaV1.2)是心脏兴奋所必需的。 收缩(EC)-偶联和通道调节失调与多种形式的心脏病有关。14-3-3 是一种普遍存在的蛋白质，它与众多细胞蛋白相互作用，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，以调节它们的运输、协同作用、磷酸化状态和/或活性。在……里面 HEK293细胞14-3-3增强了另一个电压门控钙通道(Cav2.2)的转运，并已被证明 通过与Cavβ亚基的相互作用间接改变CaV1.2的贩运，然而直接的证据和信息 关于这种调控的程度和对磷酸化的依赖，仍需进一步研究。此外，还有一些 14-3-3在心肌细胞CaV1.2通道转运/调节中的作用尚未见研究。我们致力于 这些知识在当前的应用中存在差距。自14-3-3报道以来，已为合作提供便利 门控电压依赖的心脏钠通道，NaV1.5，我们还将研究14-3-3在 CAV1.2的协同交互。当变构相互作用形成时，CaV1.2的这种门控模式发生 在簇中相邻通道的C端尾之间，使得一个通道的开口可以 与其他连接的通道通信，以提高其开放概率，并放大全细胞钙内流。 我们的研究小组先前已经证明，PKA介导的CaV1.2通道的磷酸化触发增强 这些通道运输到心室肌细胞的肌膜上，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钙电流。这有助于调整心脏 EC-联轴器，以满足战斗或逃跑时的增强需求。然而，这一分子细节增强了 走私活动尚不清楚。在这里，我们提出14-3-3在这一反应中发挥作用。我们已经确定了几个 CaV1.2和其他关键调控位点C-尾上14-3-3的推定结合位点，包括共识 PKA磷酸化位点。该项目旨在检验14-3-3调控CaV1.2贩运的假设， 导致肌膜上增强的通道聚集，从而促进协作相互作用和 放大钙离子内流。我们进一步提出，这些相互作用通过渠道得到加强 磷酸化提供了一种调节CaV1.2通道活性和EC偶联以满足需求的手段。在……里面 在这个为期两年的博士前项目中，我们将在三个具体目标上严格检验这一假设。AIM 1测试 假设14-3-3以磷酸化依赖的方式与CaV1.2相互作用。《目标2》检验了这一假设 CaV1.2的通道运输、肌膜聚集和协同作用增强了14-3-3。 目的3重点研究这种调节对心脏EC偶联的功能影响。CAV1.2频道的更改 鉴于其在心脏EC中的核心作用，贩运和调节与许多心肌病有关。 耦合；因此，我们的目标与NHLBI的使命相关。

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.