Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity.
Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity.
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DOI：
10.1016/j.yjmcc.2015.06.009
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发表时间：
2015-08
影响因子：
5
通讯作者：
Bers DM
中科院分区：
文献类型：
--
作者：
Oda T;Yang Y;Uchinoumi H;Thomas DD;Chen-Izu Y;Kato T;Yamamoto T;Yano M;Cornea RL;Bers DM
氧化应激可能导致心力衰竭 (HF) 和心律失常中的心脏兰尼碱受体 (RyR2) 功能障碍。改变的 RyR2 结构域-结构域相互作用（结构域解压缩）和钙调蛋白 (CaM) 结合亲和力是 RyR2 构象的变构耦合指标。在 HF 中，RyR2 表现出 CaM 结合减少、结构域解压缩增加和 SR Ca 泄漏增加，丹曲林可以逆转这些变化。然而，氧化应激对肌细胞 RyR2 构象和渗漏的影响尚不清楚。我们使用荧光 CaM、FKBP12.6 和结构域肽生物传感器 (F-DPc10) 直接在心肌细胞中测量：(1) 过氧化氢 (H2O2) 诱导的氧化引起的 RyR2 激活，(2) 氧化引起的 RyR2 构象变化，(3) CaM-RyR2 和 FK506 结合蛋白 (FKBP12.6)-RyR2 氧化时的相互作用，以及 (4) 丹曲林是否影响 1-3。 H2O2 用于模拟氧化应激。 H2O2 显着增加了 Ca2+ 火花和自发 Ca2+ 波的频率，而丹曲林几乎完全阻止了这些效应。 H2O2 预处理显着降低 CaM-RyR2 结合，但对 FKBP12.6-RyR2 结合没有影响。丹曲林恢复了 CaM-RyR2 结合，但对细胞内和 RyR2 氧化水平没有影响。 H2O2 还加速了 F-DPc10-RyR2 结合，而丹曲林则减缓了它的结合。因此，H2O2 会引起与 Ca 泄漏相关的构象变化（通过 CaM 和 DPc10 结合感知），而丹曲林则可逆转这些 RyR2 效应。总之，在心肌细胞中，H2O2 处理显着降低 CaM-RyR2 亲和力，对 FKBP12.6-RyR2 亲和力没有影响，并导致结构域解压缩。 Dantrolene 可以纠正结构域解压缩，恢复 CaM-RyR2 亲和力，并安静病理性 RyR2 通道门控。 F-DPc10 和 CaM 是病理生理 RyR2 状态的有用生物传感器。
Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.