环gmp依赖性蛋白激酶G对心脏良诺定受体功能的调控

IF 2.1 Q3 PHYSIOLOGY
Luis A. Gonano , Hamish M. Aitken-Buck , Akash D. Chakraborty , Luke P.I. Worthington , Tanya R. Cully , Regis R. Lamberts , Martin G. Vila-Petroff , Peter P. Jones
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引用次数: 4

摘要

cgmp依赖性蛋白激酶G (PKG)在体外磷酸化心脏ryanodine受体(RyR2)。我们的目的是确定内源性PKG的调节是否会改变RyR2介导的自发Ca2+释放,以及这种影响是否与RyR2磷酸化的变化有关。结果:用cGMP类似物8-Br-cGMP (100 μM)处理RyR2诱导表达的人胚胎肾(HEK293)细胞,激活内源性PKG,在转染腔内Ca2+传感器D1ER的细胞中,PKG的激活显著降低了RyR2介导的自发Ca2+释放的阈值(93.9±0.4%与8-Br-cGMP相比,91.7±0.8%,P = 0.04)。PKG磷酸化位点S2808和S2030的突变,无论是单独的还是联合的,都阻止了内源性PKG激活引起的Ca2+释放阈值的降低。有趣的是,尽管RyR2磷酸化位点的表达具有功能依赖性,但8-Br-cGMP激活PKG并没有促进S2808磷酸化的可检测变化(P = 0.9)。矛盾的是,KT 5823 (1 μM)对PKG的药理学抑制也降低了通过RyR2自发释放Ca2+的阈值,而不影响S2808的磷酸化。沉默RNA敲低内源性PKG表达对RyR2 S2808磷酸化也没有可量化的影响(P = 0.9)。然而,与kt5823抑制PKG不同,PKG敲低并不改变自发Ca2+释放倾向或腔内Ca2+处理。在完整的细胞模型中,内源性PKG的激活以依赖于RyR2磷酸化位点S2808和S2030的方式降低了RyR2介导的自发Ca2+释放的门槛。本研究阐明了内源性PKG对RyR2 Ca2+释放的调控,并在功能上暗示了RyR2磷酸化的作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca2+ 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 Ca2+ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca2+ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca2+ 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 (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca2+ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca2+ release propensity or luminal Ca2+ handling.

Conclusion

In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca2+ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca2+ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.

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CiteScore
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