Maximum Phosphorylation of the Cardiac Ryanodine Receptor at Serine-2809 by Protein Kinase A Produces Unique Modifications to Channel Gating and Conductance Not Observed at Lower Levels of Phosphorylation

Abstract

Abstract-It is suggested that protein kinase A (PKA)-dependent phosphorylation of cardiac ryanodine receptors (RyR2) is linked to the development of heart failure and the generation of fatal cardiac arrhythmias. It is also suggested that RyR2 is phosphorylated to 75% of maximum levels in heart failure resulting in leaky, unregulated channels gating in subconductance states. We now demonstrate that this is unlikely, as RyR2 isolated from nonfailing cardiac muscle is phosphorylated to 75% of maximum at serine-2809, and in this situation, RyR2 displays low open probability (P o ) (0.059Ϯ0.010 [SEM]; nϭ30) and normal regulation of gating by Ca 2ϩ and other ligands. However, when serine-2809 is PKA phosphorylated to maximum levels on RyR2, unique changes in channel behavior are observed. The channel displays enhanced single-channel conductance, very long open states causing large increases in P o , and no evidence of subconductance states. Dephosphorylation of channels by protein phosphatase 1 (from 75% to near 0% at serine-2809) also enhances RyR2 channel activity through abbreviation of closed lifetimes. We propose that channels phosphorylated to 75% of maximum at serine-2809 occupy a natural low point in the RyR2 activity landscape. This optimizes channel control, which can be accomplished either by enhanced or decreased phosphorylation, making the channel particularly sensitive to the kinase:phosphatase balance. Pathological situations such as heart failure might upset this balance and thereby permit prolonged stoichiometric phosphorylation of serine-2809, which would be required for dysregulation of SR Ca 2ϩ release. 7 One model of RyR2 regulation by phosphorylation of serine-2809 has dominated discussions recently and has become something of a paradigm. This model suggests that hyperphosphorylation of RyR2 on serine-2809 (defined as 3 subunits per tetramer phosphorylated at serine-2809 1 ) occurs in heart failure and is a likely cause of the cardiac dysfunction through the pathological phenotype of hyperphosphorylated RyR2 channels. This phenotype is characterized by an enhanced P o and deranged control of channel function (subconductance states and loss of coupled gating 9 ). These results led to the suggestion that hyperphosphorylation of RyR2 at PKA sites can lead to severe defects in excitation contraction (EC) coupling and the generation of fatal arrhythmias. 1,2 Other investigators, however, have failed to support this model, observing no alteration in serine-2809 phosphorylation in heart failure. 4,5 In view of the controversy surrounding the molecular basis of heart failure, we have investigated the effects of PKAdependent phosphorylation on the gating and conduction of RyR2 and related the changes in single-channel function to serine-2809 phosphorylation levels. This study has defined clear functional consequences of RyR2 phosphorylation and evaluated whether serine-2809 phosphorylation is a worthwhile marker for these functional states. We show that PKA-dependent phosphorylation of serine-2809 on RyR...

β-Adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca 2+ channel

“…This indicates a direct, SR contentindependent effect of ␤AR signaling to amplify I spark . Given that the Ca 2ϩ conductance of single RyRs is not sensitive to phosphorylation status (17,19,20), the increased I spark suggests that ␤AR stimulation synchronizes the activation of additional RyRs during a Ca 2ϩ spark. (1).…”

“…Our results show that single LCCs, in the context of a reduced coupling latency and increased coupling fidelity, increase spark amplitude in response to ␤AR stimulation. Because the Ca 2ϩ conductance of single RyRs is not sensitive to RyR phosphorylation status (17,19,20), increase in spark amplitude reflects a synchronization of RyR activation. In other words, ␤AR stimulation increases the number of RyRs that respond nearly simultaneously to the current of a single LCC.…”

“…Because RyRs are inaccessible to direct electrophysiological measurements, the ␤AR-PKA modulation of RyRs in prior studies has been investigated by different methods, yielding controversial results. In lipid bilayer experiments, the most straightforward method to measure channel behavior, RyR open probability, has been shown to increase (13,14,19,20), decrease (13,18,21), or not change (22) after PKA-mediated phosphorylation. Of note, experiments applying a rapid increase of cis(cytosolic)-side Ca 2ϩ (13) and those with millimolar trans(luminal)-side Ca 2ϩ (14,19,20) always exhibit PKA-dependent enhancement of RyR activity, agreeing well with our results.…”

“…Because RyRs are intracellular channels, they are inaccessible to direct electrophysiological measurements in intact cells. Measurements of RyR activation ex vivo in lipid bilayers have not solved this problem (13,14,(17)(18)(19)(20)(21)(22), because these measurements are controversial and inconsistent with in situ RyR behavior (23).Loose-patch confocal imaging of LCC Ca 2ϩ influx and RyR Ca 2ϩ release has demonstrated that, when triggered by the Ca 2ϩ influx from a single LCC, i.e., a Ca 2ϩ sparklet (8), a Ca 2ϩ spark represents the activation of one or a few RyRs within the same couplon (23). Confocal imaging of sparklet-spark coupling, therefore, provides a unique model for examining the RyR response to its native activator at the molecular level.…”

“…Because RyRs are intracellular channels, they are inaccessible to direct electrophysiological measurements in intact cells. Measurements of RyR activation ex vivo in lipid bilayers have not solved this problem (13,14,(17)(18)(19)(20)(21)(22), because these measurements are controversial and inconsistent with in situ RyR behavior (23).…”