Published online before print May 18, 2006, doi: 10.1161/01.RES.0000227506.43292.df
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© 2006 American Heart Association, Inc.
Cellular Biology |
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
From the Department of Pharmacology (S.C., R.S.), University of Bristol; and Institute of Membrane & Systems Biology (J.C.), University of Leeds, UK.
Correspondence to Rebecca Sitsapesan, University of Bristol, Department of Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. E-mail r.sitsapesan{at}bris.ac.uk
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 (Po) (0.059±0.010 [SEM]; n=30) and normal regulation of gating by Ca2+ 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 Po, 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 Ca2+ release.
Key Words: phosphorylation • ryanodine receptor • Ca2+ release • excitation–contraction coupling
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