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

阐明 L 型钙通道 (CaV1.2) 中 β-肾上腺素能调节的机制

• 批准号: 10490970
• 负责人: Arianne Papa
• 金额: $ 0.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490970
• 关键词: Adrenergic Agents; Adrenergic beta-Agonists; Alanine; Arrhythmia; Attenuated; Ca(2+)-Transporting ATPase; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Cells; Characteristics; Cyclic AMP-Dependent Protein Kinases; Data; Electrophysiology (science); Enzymes; Exercise; Exons; Forskolin; Goals; Heart; Heart Atrium; Heart Hypertrophy; Heart failure; In Vitro; Investigation; Knock-in; Knock-in Mouse; L-Type Calcium Channels; Label; Laboratories; Lead; Link; Macromolecular Complexes; Mass Spectrum Analysis; Measurement; Mediating; Methods; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Pacemakers; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiology; Play; Proteomics; Regulation; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Sinus; Surface; Sympathetic Nervous System; System; Techniques; Testing; base; calmodulin-dependent protein kinase II; design; experience; experimental study; fighting; heart function; hormone regulation; in vivo; inhibitor; mouse model; mutant; novel; novel strategies; phospholamban; prevent; protein activation; reconstitution; response; skills; trafficking; voltage

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.

项目总结