L-type channel trafficking and modulation in heart

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SUMMARY Ca2+ influx through voltage-dependent L-type (CaV1.2) channels into cardiomyocytes is a multi-dimensional signal that mediates excitation-contraction (E-C) coupling, controls action potential duration, and regulates gene expression. Dysregulation of CaV1.2 causes heart disease, the leading cause of death in the US and worldwide. β-adrenergic up-regulation of CaV1.2 underlies the positive inotropic response essential for the fight or flight response. Despite decades of intense focus, the precise molecular mechanisms underlying β-adrenergic up- regulation of Ca2+ influx via CaV1.2 in cardiomyocytes remained elusive and controversial. In the last funding period, using an ascorbate-peroxidase (APEX2)-proximity-labeling method, we discovered that under basal conditions the Ca2+ channel inhibitor Rad, a small G-protein, is enriched near CaV1.2 but is depleted upon exposure to a β-adrenergic agonist. Our findings fundamentally recast the mechanism for β-adrenergic regulation of CaV1.2: under basal conditions, Rad inhibits a sub-population of CaV1.2 in cardiomyocytes; upon adrenergic activation, protein kinase A (PKA) phosphorylates Rad and relieves this inhibition to increase whole- cell L-type current (ICa,L). The implications of this new model are profound and raise a slew of fundamental questions that we are uniquely poised to address based on several innovative approaches we have developed and insights we have attained in the last funding cycle. This renewal application is motivated by the overall hypotheses that deepened understanding of the precise mechanisms underlying kinase regulation of CaV1.2 via Rad will not only provide fundamental new insights into cardiac physiology in the fight or flight response and beyond, but may also be exploited to develop novel positive inotropic agents to combat heart disease. We combine innovative approaches developed in the Marx (proximity labeling proteomics, knockin mouse models) and Colecraft (nanobody-based targeted regulation of CaV1.2) labs to address three specific Aims. 1) Elucidate mechanisms and determinants underlying Rad targeting to CaV1.2 in heart and β-adrenergic regulation of ICa,L. 2) Elucidate mechanisms and physiological role of kinase-phosphatase balance regulation of basal CaV1.2 in cardiomyocytes. 3) Engineer the kinase-phosphatase balance in the CaV1.2 nanodomain using targeted recruitment of kinases.