Atrial fibrillation (AF) is the most common cardiac arrhythmia and accounts for substantial morbidity and mortality. In atrial cardiomyocytes, excitation-contraction (E-C) coupling is initiated by activation of voltage-gated Na+ channels, NaV1.5. Depolarization of the membrane potential, by Na+ channels, leads to activation of voltage- gated Ca2+ channels, thereby triggering Ca2+-induced Ca2+ release in atria cardiomyocytes. During the prior funding period, we developed innovative methods to probe determinants triggering persistent Na+ current- induced spontaneous AF in mice. We identified heterogeneously prolonged action potential duration, abnormal Ca2+ handling, increased reactive oxygen species and oxidation of ryanodine receptors (RyR2) as drivers of atrial cardiomyopathy and arrhythmias. In this renewal, we will expand these studies, now applying innovative proximity labeling, novel mouse models, and groundbreaking atomic resolution structural studies of the human recombinant RyR2 channel. Three Aims are proposed: (1) Determine the role of adrenergic regulation of Ca2+ channels in atrial E-C coupling and arrhythmogenesis. Recently, we identified the mechanism by which β- adrenergic agonists stimulate voltage-gated Ca2+ channels. 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 residues on Rad relieves constitutive inhibition of CaV1.2. To determine the role of PKA-induced stimulation of Ca2+ currents in the atria, we will use knock-in mice with the four PKA phosphorylation sites of Rad mutated to alanine, mice lacking the Rad-β subunit interaction as well mice expressing RyR2 channels that cannot be phosphorylated by PKA. Using these mice, we will determine whether phosphorylation of Rad and/or phosphorylation of RyR2 are required for adrenergic agonist-induced AF. (2) To define the atrial NaV1.5, CaV1.2 and RyR2 interactomes and “neighborhoods” in atrial cardiomyocytes under physiological and pathological conditions. We propose to use proximity labeling approaches to compare the neighborhoods of Ca2+, Na+ and RyR2 channels in the atria, and determine how these neighborhoods change in pathological conditions, such as HF, which predisposes patients to AF. (3) To elucidate the role of oxidation of RyR2 in the pathogenesis of AF. We speculate that oxidation of RyR2 and the resultant SR Ca2+ leak is an essential downstream effector leading to atrial arrhythmias. We will identify which cysteine residues are oxidized in atrial RyR2 in both mice and humans with AF. The structural effects of the identified oxidized cysteine residues will be investigated using an atomic-resolution structure of human recombinant RyR2 determined by cryo-EM, and by electrophysiological studies of heterologously expressed mutant RyR2 channels. Taken together, these highly innovative and novel studies will elucidate...