L-type channel trafficking and modulation in heart

摘要 钙离子通过电压依赖性L型(CaV1.2)通道内流到心肌细胞是一种多维的 介导兴奋-收缩(E-C)偶联、控制动作电位时程和调节基因的信号 表情。CaV1.2的失调会导致心脏病，这是美国和世界范围内的主要死亡原因。 β-肾上腺素能上调CaV1.2是战斗或逃跑所必需的正性肌力反应的基础 回应。尽管几十年来人们高度关注，但β-肾上腺素能上调-的确切分子机制- 通过CaV1.2对心肌细胞内钙内流的调节仍然是难以捉摸和有争议的。在上一笔资金中 期间，使用抗坏血酸-过氧化物酶(APEX2)-邻近标记方法，我们发现在基础 条件：钙通道抑制物Rad，一种小G蛋白，在CaV1.2附近富含，但在CaV1.2附近被耗尽 接触β肾上腺素能激动剂。我们的发现从根本上重塑了β-肾上腺素能 CaV1.2的调节：在基础条件下，Rad抑制心肌细胞中CaV1.2的一个亚群； 肾上腺素能激活，蛋白激酶A(PKA)磷酸化Rad，并解除这种抑制，以增加整体- 细胞L型电流(ICa，L)。这一新模式的影响是深远的，并提出了一系列基本的 根据我们开发的几种创新方法，我们独特地准备解决的问题 以及我们在上一个资金周期中获得的见解。此次续签申请的动机是 假说加深了对CaV1.2激酶调控的精确机制的理解 RAD不仅将为心脏生理学在战斗或逃跑反应中提供基本的新见解，而且 不仅如此，还可能被利用来开发新的正性肌力药物来对抗心脏病。我们 结合马克思发展的创新方法(邻近标记蛋白质组学、敲击小鼠模型) 和Colraft(基于纳米机构的CaV1.2靶向调控)实验室，以解决三个具体目标。1)澄清 Rad靶向心脏CaV1.2的机制和决定因素及β对ICa的肾上腺素能调节 2)阐明了基础CaV1.2的激酶-磷酸酶平衡调节机制及其生理作用。 心肌细胞。3)使用靶向技术在CaV1.2纳米结构域中设计激酶-磷酸酶平衡 激活酶的招募。

SUMMARY Ca2+ influx through voltage-dependent L-type (CaV1.2) channels into cardiomyocytes is a multi-dimensional signal that mediates excitation-contraction (E-C) coupling, controls action potential duration, and regulates gene expression. Dysregulation of CaV1.2 causes heart disease, the leading cause of death in the US and worldwide. β-adrenergic up-regulation of CaV1.2 underlies the positive inotropic response essential for the fight or flight response. Despite decades of intense focus, the precise molecular mechanisms underlying β-adrenergic up- regulation of Ca2+ influx via CaV1.2 in cardiomyocytes remained elusive and controversial. In the last funding period, using an ascorbate-peroxidase (APEX2)-proximity-labeling method, we discovered that under basal conditions the Ca2+ channel inhibitor Rad, a small G-protein, is enriched near CaV1.2 but is depleted upon exposure to a β-adrenergic agonist. Our findings fundamentally recast the mechanism for β-adrenergic regulation of CaV1.2: under basal conditions, Rad inhibits a sub-population of CaV1.2 in cardiomyocytes; upon adrenergic activation, protein kinase A (PKA) phosphorylates Rad and relieves this inhibition to increase whole- cell L-type current (ICa,L). The implications of this new model are profound and raise a slew of fundamental questions that we are uniquely poised to address based on several innovative approaches we have developed and insights we have attained in the last funding cycle. This renewal application is motivated by the overall hypotheses that deepened understanding of the precise mechanisms underlying kinase regulation of CaV1.2 via Rad will not only provide fundamental new insights into cardiac physiology in the fight or flight response and beyond, but may also be exploited to develop novel positive inotropic agents to combat heart disease. We combine innovative approaches developed in the Marx (proximity labeling proteomics, knockin mouse models) and Colecraft (nanobody-based targeted regulation of CaV1.2) labs to address three specific Aims. 1) Elucidate mechanisms and determinants underlying Rad targeting to CaV1.2 in heart and β-adrenergic regulation of ICa,L. 2) Elucidate mechanisms and physiological role of kinase-phosphatase balance regulation of basal CaV1.2 in cardiomyocytes. 3) Engineer the kinase-phosphatase balance in the CaV1.2 nanodomain using targeted recruitment of kinases.