Investigating Cardiac Ion Channels by Novel Methods

我们的长期总体目标是发现潜在的生理动态平衡调节机制 CaV1.2在心脏中的通道，并确定心力衰竭和心律失常的新治疗靶点。CAV1.2， L钙通道在心肌兴奋收缩偶联中起关键作用，是心肌细胞内钙离子通道的重要靶点。 交感神经系统和几条信号通路。在战斗中增加心脏的收缩能力-否则- 飞行反应是由CaV1.2通道的β-肾上腺素能增强引起的。在转基因小鼠心脏中 完全表达PKA磷酸化位点缺陷突变体CaV1.2CaV1.2α1C和β亚基，这一调控持续存在， 暗示有渠道外因素的参与。最近，我们发现了β-肾上腺素能 激动剂刺激电压门控钙通道。我们表达了与抗坏血酸结合的α1C或β2B亚基。 并使用多重定量蛋白质组学方法追踪小鼠心脏中的数百种蛋白质 距离CaV1.2很近。我们观察到，钙通道抑制剂Rad，一种单体G蛋白，是 在CaV1.2微环境中丰富，但在β-肾上腺素能刺激过程中被耗尽。PKA催化 Rad上特定的丝氨酸残基的磷酸化降低了其与辅助β亚基的亲和力，并缓解了 观察到CaV1.2的结构性抑制随着通道开放概率的增加而增加。我们提出三个目标： (1)利用Rad的四个PKA磷酸化位点突变为丙氨酸的敲入小鼠，以及 不能结合Rad的突变型CAVβ亚单位的心脏特异表达，我们将在 心肌细胞Rad磷酸化在体内调节心肌收缩能力中的作用。(二)有 成功地应用邻近标记，我们现在还提出了鉴定A-激酶锚定蛋白 (AKAP)促进心肌细胞CaV1.2的β肾上腺素能调节。AKAP的身份 β对心肌细胞CaV1.2的肾上腺素能调节作用尚不清楚。(3)cGMP激活PKG 抑制CaV1.2并拮抗β-肾上腺素能对心肌细胞钙电流的刺激。战略PKG 因此，激活可以作为CaV1.2及其伴随的肾上腺素能刺激的靶向抑制因子 心律不齐。我们推测，PKG信号阻断β肾上腺素能诱导的CaV1.2的刺激至少是通过 几种机制之一：i)通过直接PKG磷酸化α1C或β2B；ii)通过防止招募 PKA到CaV1.2复合体；iii)防止Rad从心脏中的CaV1.2复合体解离。至 评估是否需要α1C或β2B的PKG磷酸化，我们将利用我们的完全磷酸化突变的α1C和 β2B转基因小鼠具有正常β肾上腺素能刺激的CaV1.2。剖析上行信令 途径，我们将利用邻近蛋白质组学。三个目标，这将提供关键的新理解 关于心肌细胞内钙内流的调节，与理解 心肌收缩能力调节和心律失常发生的分子机制。

Our long-term overall goals are to discover physiologic homeostatic mechanisms underlying regulation of CaV1.2 channels in the heart and to identify novel therapeutic targets for heart failure and arrhythmias. CaV1.2, the L-type Ca2+ channel that plays a key role in cardiac excitation-contraction coupling, is an important target of the sympathetic nervous system and several signaling pathways. Increased cardiac contractility during fight-or- flight response is caused by β-adrenergic augmentation of CaV1.2 channels. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant CaV1.2 α1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Recently, we identified the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed α1C or β2B subunits conjugated to ascorbate- peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in close proximity to CaV1.2. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-subunits and relieves constitutive inhibition of CaV1.2 observed as an increase in channel open probability. We propose three Aims: (1) Using knock-in mice with the four PKA phosphorylation sites of Rad mutated to alanine, and mice with cardiac-specific expression of a mutant CaVβ subunit that cannot bind Rad, we will determine in cardiomyocytes the role of Rad phosphorylation in regulating cardiac contractility in vivo. (2) Having successfully applied proximity labeling, we now also propose to identify the A-kinase anchoring proteins (AKAPs) that facilitate β-adrenergic regulation of CaV1.2 in cardiomyocytes. The identity of the AKAP that facilitates β-adrenergic regulation of CaV1.2 in cardiomyocytes is unknown. (3) PKG activation by cGMP inhibits CaV1.2 and counteracts β-adrenergic stimulation of Ca2+ current in cardiomyocytes. Strategic PKG activation could therefore serve as a targeted suppressor of adrenergic stimulation of CaV1.2 and concomitant arrhythmias. We hypothesize that PKG signaling blocks β-adrenergic-induced stimulation of CaV1.2 by at least one of several mechanisms: i) by direct PKG phosphorylation of α1C or β2B; ii) by preventing the recruitment of PKA to the CaV1.2 complex; iii) by preventing the dissociation of Rad from the CaV1.2 complex in the heart. To assess whether PKG phosphorylation of α1C or β2B is required, we will utilize our fully phospho-mutant α1C and β2B transgenic mice that have normal β-adrenergic stimulation of CaV1.2. To dissect the upstream signaling pathways, we will utilize proximity proteomics. The three Aims, which will provide key new understandings concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding the molecular mechanisms responsible for the modulation of cardiac contractility and arrhythmogenesis.