Phosphorylation Activates RyR2 By Uncoupling The Channel From The Influence Of Cytosolic Ca2+ But Also Inhibits RyR2 By A Distinct Mechanism

Abstract

Phosphorylation of RyR2 is generally thought to be associated with an increase in open probability (Po), however, there is disagreement over which phosphorylation sites are responsible. To investigate the gating mechanisms underlying phosphorylation-induced changes in RyR2 Po, we incorporated native sheep RyR2 into bilayers under voltage-clamp conditions with luminal Ca2+ as the permeant ion. Phosphorylation of RyR2 channels above basal levels, either by PKA or by activation of an endogenous kinase (by incubation with Mg2+ATP) leads to massive increases in Po that are associated with very long open states. The channels become effectively uncoupled from the influence of cytosolic Ca2+ because reductions in cytosolic [Ca2+] to sub-activating levels do not reduce open lifetimes nor significantly reduce Po. Treatment of channels with the phosphatase, PP1 reverses these gating changes returning the channels to a cytosolic Ca2+-sensitive mode of gating. In contrast, to the gating changes described above which are specific for phosphorylation of RyR2 above basal levels, complete dephosphorylation of RyR2 by PP1 treatment causes significant increases in Po. Po increased from 0.074±0.032 to 0.218±0.028 (SEM;n=4) after incubation with PP1 (5 units). The channel gating associated with completely dephosphorylated channels, or channels phosphorylated to 75% of maximum at serine-2809, demonstrates that the channels are sensitive to cytosolic [Ca2+]. We cannot yet assign specific phosphorylation sites to particular change in channel gating but it is clear that phosphorylation of RyR2 exerts effects which span the entire range of the RyR2 activity landscape. The huge scope for changing RyR2 channel activity suggests that phosphorylation of RyR2 is important for physiological regulation of the channel and that dysregulation of SR Ca2+-release would be expected with prolonged hyperphosphorylation.Supported by the BHF