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

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.