The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Cat2+ leak in ventricular myocytes
The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Cat2+ leak in ventricular myocytes
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DOI：
10.1016/j.yjmcc.2017.01.015
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发表时间：
2017-03-01
影响因子：
5
通讯作者：
Zima, Aleksey V.
中科院分区：
文献类型：
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作者：
Bovo, Elisa;Huke, Sabine;Zima, Aleksey V.
蛋白激酶A（PKA）对心肌Ryanodine受体（2型RyR或RyR 2）磷酸化的功能影响仍然存在很大争议。在这项研究中，我们的特点是PICA介导的RyR 2磷酸化水平和肌浆网（SR）的Ca 2+释放和泄漏透化兔心室肌细胞之间的功能联系。分别用Fluo-4和Fluo-5 N测量胞浆[Ca 2 +]和SR内[Ca 2 +]（SR）的变化。RyR 2磷酸化的变化在两个PICA网站，丝氨酸-2031和2809，测量磷酸化特异性抗体。cAMP（10 μ M）使Ca ~（2+）火花频率增加约2倍。这种效应与SR Ca 2+负荷从0.84增加到1.24 mM有关。PICA抑制肽（PKI; 10 μ M）消除了SR Ca 2+负荷和火花频率的cAMP依赖性增加。当SERCA被毒胡萝卜素完全阻断时，cAMP不影响RyR 2介导的Ca ~（2+）泄漏。cAMP对RyR 2功能的影响的缺乏可以通过肌膜透化后RyR 2在丝氨酸-2809处的几乎最大磷酸化来解释。这种高RyR 2磷酸化水平可能是透化后蛋白激酶和磷酸酶活性之间平衡转变的结果。当使用激酶抑制剂星形孢菌素将丝氨酸-2809处的RyR 2磷酸化降低至其“基础”水平（即完整肌细胞中的RyR 2磷酸化水平）时，SR Ca 2+渗漏显著降低。令人惊讶的是，蛋白磷酸酶1（PP 1）的RyR 2的进一步去磷酸化显着增加SR Ca 2+泄漏。同时，在相同的实验条件下，RyR 2在丝氨酸2031处的磷酸化没有显著变化。这些结果表明，PICA磷酸化RyR 2对心室肌细胞SR Ca 2+渗漏具有复杂的影响。在RyR 2磷酸化的中间水平，SR Ca 2+泄漏是最小的。然而，RyR 2的完全去磷酸化和最大磷酸化会增加SR Ca 2+渗漏。(C)2017爱思唯尔有限公司版权所有
Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PICA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+](SR) were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PICA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10 mu M) increased Ca2+ spark frequency approximately two -fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24 mM. PICA inhibitory peptide (PKI; 10 mu M) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PICA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak. (C) 2017 Elsevier Ltd. All rights reserved.