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

项目摘要 我的总体目标是揭示负责电压门控的生理调节机制， 心脏中的钙通道。通过电压门控L-型钙通道（CaV1.2）的钙内流是一种抑制钙内流的机制。 启动每个心跳的基本信号。功能失调的钙通道运输和调节已经被 与心律失常、心脏肥大和心力衰竭的机制有关。在“战斗或逃跑”期间 蛋白激酶A（PKA）的β-肾上腺素能激活增加了这种钙内流， 心肌收缩力尽管经过数十年的研究，该途径激活的详细机制 心脏中的钙通道仍然未知。实验室的初步数据表明， 钙通道抑制剂Rad是一种小G蛋白，是PKA调节CaV1.2的缺失环节。 基于邻近蛋白质组学，Rad在CaV1.2微环境中富集，但在β- 肾上腺素能刺激心脏我们证实在异源表达系统中，Rad共表达 在全细胞水平上完全重建PKA调节，并重现PKA的单通道特征 调变此外，我们证明了Rad也是PKA磷酸化的关键功能靶标， 因为消除Rad磷酸化位点消除了毛喉素介导的钙通道刺激。在 最后，潜在的机制被证明是简单而优雅的-基线的Rad抑制CaV1.2活性， 而Rad的PKA磷酸化解除这种抑制。我的假设是拉德的磷酸化 对于心脏中的钙通道调节，以及钙通道的β-肾上腺素能调节的丧失 减弱肾上腺素能诱导的变力性增加。 β-肾上腺素能调节的重要组成部分， 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的爆震- 需要细胞电生理学技术和心脏功能的体内测量。的 两个目标将确定负责心肌细胞钙流入调节的新机制，这可能 导致调节心脏收缩力和心律失常新方法。

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.