REGULATION OF CARDIAC ION CHANNELS VIA STRESS SIGNALING

BACKGROUND Stress signaling typically involves stimulation of the β-adrenergic pathway which leads to the subsequent activation of cAMP-dependent protein kinase A (PKA). PKA has thus been proposed to regulate calcium entry into cardiomyocytes during stress signaling via the L-type calcium channel (CaV1.2) and the ryanodine receptor 2 (RyR2). Ryanodine Receptors (RyRs) are huge intracellular ion channels, located in the sarcoplasmic and endoplasmic reticulum, where they control the release of stored calcium ions whereas CaV1.2 channel is located on the plasma membrane. Both channels are targeted by disease-causing mutations and are under strict regulation by auxiliary proteins, small molecules, and post-translational modifications (PTMs). PTMs of these channels is a cornerstone of their physiological and pathophysiological regulation; and aberrant phosphorylation has been implicated in a multitude of disorders ranging from heart failure to Alzheimer's disease. Despite decades of research, the molecular mechanism underlying this modulation is enigmatic with multiple sites being implicated. Further there is some controversy regarding which sites are being phosphorylated by PKA and which are important for β-adrenergic responses. METHODS AND RESULTS Using high-resolution structures determined via X-ray crystallography, we show how the catalytic domain of PKA engages both the RyR2 and CaV1.2 channels, highlighting the target sites that are preferentially oriented for phosphoryl transfer. Using isothermal titration calorimetry (ITC) and kinase assays, we determined the binding affinities and enzymatic activities of PKA to various RyR2 and CaV1.2 substrates. CONCLUSION RyR2 domain engages PKA by wrapping around the large-lobe of its catalytic subunit, resulting in an extensive interface not seen in PKA complexes with isolated peptides. We also trapped complexes in both closed and open forms PKA, showing that the RyR2 substrate can bind prior to closing of PKA. The interface is targeted by multiple disease-associated mutations that can affect the interaction and catalytic activity. Finally, we found when one site is phosphorylated in RyR2, another site is more likely to be phosphorylated by PKA, which can lead to amplification of the stress signal. We also determined the X-ray crystal structure of a C-terminal peptide from CaV1.2 containing S1981 in complex with PKA. We measured a dissociation constant of 35 uM toward this peptide via ITC, which is comparable to other PKA substrates. Our kinase assays corroborated this finding with strong activity towards S1981. In contrast, the affinity and enzymatic activity of PKA towards S1718 is substantially lower despite being implicated as an important PKA phosphorylation site.