Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G.

BACKGROUND

The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) . We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca release and whether this effect is linked to a change in RyR2 phosphorylation.

METHODS

& Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP,  = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation ( = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation ( = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca release propensity or luminal Ca handling.

CONCLUSION

In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.