Investigating Cardiac Ion Channels by Novel Methods

Our long-term overall goals are to discover physiologic homeostatic mechanisms underlying regulation of CaV1.2 channels in the heart and to identify novel therapeutic targets for heart failure and arrhythmias. CaV1.2, the L-type Ca2+ channel that plays a key role in cardiac excitation-contraction coupling, is an important target of

the sympathetic nervous system and several signaling pathways. Increased cardiac contractility during fight-or- flight response is caused by β-adrenergic augmentation of CaV1.2 channels. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant CaV1.2 α1C and β subunits, this regulation persists,

implying involvement of extra-channel factors. Recently, we identified the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed α1C or β2B subunits conjugated to ascorbate- peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in

close proximity to CaV1.2. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-subunits and relieves

constitutive inhibition of CaV1.2 observed as an increase in channel open probability. We propose three Aims: (1) Using knock-in mice with the four PKA phosphorylation sites of Rad mutated to alanine, and mice with cardiac-specific expression of a mutant CaVβ subunit that cannot bind Rad, we will determine in

cardiomyocytes the role of Rad phosphorylation in regulating cardiac contractility in vivo. (2) Having successfully applied proximity labeling, we now also propose to identify the A-kinase anchoring proteins (AKAPs) that facilitate β-adrenergic regulation of CaV1.2 in cardiomyocytes. The identity of the AKAP that

facilitates β-adrenergic regulation of CaV1.2 in cardiomyocytes is unknown. (3) PKG activation by cGMP inhibits CaV1.2 and counteracts β-adrenergic stimulation of Ca2+ current in cardiomyocytes. Strategic PKG activation could therefore serve as a targeted suppressor of adrenergic stimulation of CaV1.2 and concomitant

arrhythmias. We hypothesize that PKG signaling blocks β-adrenergic-induced stimulation of CaV1.2 by at least one of several mechanisms: i) by direct PKG phosphorylation of α1C or β2B; ii) by preventing the recruitment of PKA to the CaV1.2 complex; iii) by preventing the dissociation of Rad from the CaV1.2 complex in the heart. To

assess whether PKG phosphorylation of α1C or β2B is required, we will utilize our fully phospho-mutant α1C and β2B transgenic mice that have normal β-adrenergic stimulation of CaV1.2. To dissect the upstream signaling pathways, we will utilize proximity proteomics. The three Aims, which will provide key new understandings

concerning the regulation of Ca2+ influx in cardiomyocytes, are highly relevant towards understanding the molecular mechanisms responsible for the modulation of cardiac contractility and arrhythmogenesis.