Elucidating the mechanism of beta-adrenergic regulation in L-type Calcium Channels (CaV1.2)

项目摘要 我的总体目标是发现负责电压门控的生理调节的机制 心脏中的钙通道。电压门控L型钙通道(CaV1.2)的钙内流是一种 启动每个心跳的基本信号。功能障碍的钙通道转运和调节一直是 与心律失常、心肌肥厚和心力衰竭的机制有关。在《不战即逃》中 作为回应，蛋白激酶A(PKA)的β-肾上腺素能激活增加了这种钙内流，并增加了 心脏收缩能力。尽管经过了几十年的研究，但该通路激活的详细机制 心脏中的钙通道仍不清楚。实验室强劲的初步数据表明， 钙通道抑制剂Rad是一种小G蛋白，是使PKA能够调节CaV1.2的缺失环节。 根据邻近蛋白质组学，Rad在CaV1.2微环境中富含，但在Beta- 心脏中的肾上腺素能刺激。我们在异源表达系统中证实Rad共表达 全信元水平的全重构PKA调制及其单通道特性 调制。此外，我们还证明Rad也是PKA磷酸化的关键功能靶点， 随着Rad磷酸化位点的消除，Forsklin对钙通道的刺激作用也随之消失。在……里面 最终，潜在的机制被证明是简单而优雅的-基线时的Rad抑制CaV1.2的活性， 而Rad的PKA磷酸化则解除了这种抑制。我的假设是Rad的磷酸化就足够了 对于心脏钙通道的调节，以及β-肾上腺素能钙通道调节的丧失 减弱肾上腺素能引起的变力作用的增加。 β-肾上腺素能调节的基本成分， 通过两个目标，我将：(1)验证Rad磷酸化作为一种 以及(2)评估PKA诱导的刺激的贡献 钙电流在形成对β-肾上腺素能激动剂的心脏反应中的作用。这两个目标都利用了新奇的敲击- 在小鼠身上，需要细胞电生理技术和在体心脏功能测量。这个 两个目标将确定负责调节心肌细胞钙内流的新机制，这可能 导致新的方法来调节心脏收缩能力和心律失常。

Project Summary My overall goal is to uncover mechanisms responsible for physiological regulation of the voltage-gated calcium channel in the heart. Calcium influx through voltage-gated L-type calcium channels (CaV1.2) is an essential signal initiating each heartbeat. Dysfunctional calcium channel trafficking and regulation have been implicated in the mechanisms of arrhythmias, cardiac hypertrophy, and heart failure. During the “fight or flight” response, beta-adrenergic activation of protein kinase A (PKA) increases this calcium influx and increases cardiac contractility. Despite decades of investigation, the detailed mechanism by which this pathway activates calcium channels in the heart remains unknown. Strong preliminary data in the laboratory suggest that the calcium channel inhibitor Rad, a small G-protein, is the missing link that enables PKA regulation of CaV1.2. Based on proximity proteomics, Rad is enriched in the CaV1.2 microenvironment but is depleted during beta- adrenergic stimulation in the heart. We confirmed in a heterologous expression system that Rad co-expression fully-reconstituted PKA modulation at the whole-cell level and recapitulated single-channel characteristics of PKA modulation. Furthermore, we demonstrated that Rad is also the key functional target of PKA phosphorylation, as eliminating Rad phosphorylation sites abolished forskolin-mediated stimulation of the calcium channels. In the end, the underlying mechanism turns out to be simple and elegant – Rad at baseline inhibits CaV1.2 activity, while PKA phosphorylation of Rad relieves this inhibition. My hypothesis is that Rad phosphorylation is sufficient for calcium channel regulation in the heart, and that loss of beta-adrenergic regulation of calcium channels attenuates adrenergic-induced increase in inotropy. essential component of beta-adrenergic regulation, Via two Aims, I will: (1) validate Rad phosphorylation as an and (2) assess the contribution of PKA-induced stimulation of calcium currents in forming the cardiac response to beta-adrenergic agonists. Both Aims utilize novel knock- in mice, and require cellular electrophysiological techniques and in vivo measurements of cardiac function. The two Aims will identify new mechanisms responsible for regulation of calcium influx in cardiomyocytes, which may lead to novel approaches to modulate cardiac contractility and arrhythmias.