Illuminating the function regulome of cardiac L-type Ca2+ channels in health and disease

项目摘要 CaV1.2通道及其调节对于正常功能至关重要， 心脏的病理生理学CaV1.2功能和调节的中断导致电和 Ca 2+紊乱是危及生命的心脏疾病（包括心律失常）的基础， 心肌病和心力衰竭。CaV1.2作为Ca 2+流入的主要门户， 启动兴奋-收缩偶联的心肌细胞， 形态学，基础肌力，并支持活性依赖性基因转录。履行 由于细胞功能多样，CaV1.2通道募集了丰富的调节蛋白， 多相大分子复合物在这方面，CaV1.2辅助亚基（α2δ和α 2 δ） β2）和各种调节蛋白已成为协调贩运的关键参与者， 门控和肾上腺素能调节。最近的工作使用邻近蛋白质组学表明， CaV1.2邻域延伸超出这些充分研究的亚基。然而，非经典的 相互作用的蛋白质调节CaV1.2功能，以及通道调节的改变如何有助于 病理生理学在很大程度上是未知的。这一差距阻碍了我们在理解 CaV1.2在心脏生理学中的作用，并阻碍了新疗法的发展。因此， 该项目在PPG内的目标是构建下一代功能蓝图， 相关的心脏CaV1.2调节剂，并确定通道误调节的贡献 在心力衰竭中的作用。我们开发了一种新的功能筛选方法 以系统地鉴定潜在的CaV1.2调节剂。我们进一步开展广泛的 两种非经典CaV1.2调节剂的机制分析― ―富含亮氨酸重复序列 蛋白质10（Lrrc 10）和Junctophilin-2（Jph 2），两者都与心肌病和心力衰竭有关。 此外，我们利用巴基斯坦基因组资源，提供基因组测序 数据和深心脏代谢表型的个人与功能缺失突变，在这两个 Lrrc 10和Jph 2的功能，以辨别蛋白质对人体生理学的功能重要性。三 具体目的是：（1）鉴定CaV1.2的功能相关的非典型调节剂。（二） 阐明Lrrc 10调节CaV1.2的机制和DCM连锁突变的影响。 (3)确定Jph 2调节CaV1.2的机制和人类突变的影响。

PROJECT SUMMARY CaV1.2 channels and their modulation are fundamentally important for the normal function and pathophysiology of the heart. Disruption of CaV1.2 function and regulation results in electrical and Ca2+ disturbances that underlie life-threatening cardiac diseases including arrhythmias, cardiomyopathies, and heart failure. CaV1.2 serves as the primary portal for Ca2+ influx into cardiomyocytes that initiates excitation-contraction coupling, sculpts the action potential morphology, underlies inotropy, and supports activity-dependent gene transcription. To fulfill diverse cellular functions, CaV1.2 channels recruit a rich repertoire of regulatory proteins, yielding heterogeneous macromolecular complexes. In this regard, the CaV1.2 auxiliary subunits (α2δ and β2) and various regulatory proteins have emerged as key players in orchestrating trafficking, gating, and adrenergic regulation. Recent work using proximity proteomics suggests that the CaV1.2 neighborhood extends beyond these well-studied subunits. Yet, how the non-canonical interacting proteins tune CaV1.2 function, and how alterations in channel regulation contribute to pathophysiology are largely unknown. This gap impairs forward progress in our understanding of CaV1.2 in cardiac physiology and thwarts the development of new therapies. Thus, the overall objective of this project within the PPG is to construct a next-generation blueprint of functionally- relevant cardiac CaV1.2 modulators, and to determine the contribution of channel mis-regulation by non-canonical regulators in heart failure. We develop a novel functional screening approach to systematically identify potential CaV1.2 modulators. We further undertake extensive mechanistic analysis of two non-canonical CaV1.2 modulators―Leucine rich repeat containing protein 10 (Lrrc10) and Junctophilin-2 (Jph2), both linked to cardiomyopathies and heart failure. In addition, we leverage the Pakistan Genome Resource which provides genome sequencing data and deep cardio-metabolic phenotyping of individuals with loss-of-function mutations in both Lrrc10 and Jph2 to discern the function importance of the proteins to human physiology. The three specific aims are: (1) Identify functionally-relevant non-canonical modulators of CaV1.2. (2) Elucidate the mechanism of Lrrc10 modulation of CaV1.2 and effects of DCM-linked mutations. (3) Identify mechanism of Jph2 modulation of CaV1.2 and effects of human mutations.