beta-adrenergic modulation of cardiac ryanodine receptor

描述(申请人提供)：在心脏，少量的钙离子通过L类钙离子通道(DHPR)内流触发兰尼定受体(RyRs)的开放，然后RyR通过钙离子诱导的钙释放(CICR)过程从肌浆网(SR)释放大量钙离子。因此，心脏收缩的力量在很大程度上依赖于RyRs释放的钙离子的大小；在应激期间或在运动环境中，对心室肌细胞的β-肾上腺素能刺激增加了钙释放，从而增加了收缩的力量。β-肾上腺素能刺激通过激活PKA使磷蛋白磷酸化而增加钙释放，从而解除其对肌浆网钙泵(SERCA)的抑制，增加肌浆网钙负荷。较高的肌浆网钙负荷会导致较高的钙释放。RyRs的大量磷酸化也见于β-肾上腺素能刺激，但RyR磷酸化在正常和疾病状态下的作用仍然存在争议。这项建议试图确定RyR磷酸化的生理影响，参与这一反应的分子参与者，以及它对心力衰竭的影响。结合抗RyRs的磷酸化特异性抗体、全心β-肾上腺素能刺激、细胞钙成像和准生理条件下单个RyR活性的记录，我们提出：1)确定由β-肾上腺素能刺激激活的使RyRs磷酸化的蛋白激酶(S)，它们在RyR序列中的特异性磷酸化位置(S)，以及它们在生理条件下的参与程度。2)通过直接记录野生型和突变型RyR通道在准生理条件下的活性，确定RyR中特定磷酸化位点的功能输出。3)探讨RyR磷酸化及其对应反应RyR去磷酸化在心力衰竭钝性钙释放的发病机制中的作用。这些研究可能为控制RyR磷酸化的机制以及这一过程对心肌细胞钙稳态的功能后果提供新的、新的见解。因此，它们可能有助于解决RyR磷酸化在心力衰竭发病机制中的有争议的作用。

DESCRIPTION (provided by applicant): In the heart, a small influx of Ca2+ through L-type Ca2+ channels (DHPRs) triggers the opening of ryanodine receptors (RyRs), which in turn release massive amounts of Ca2+ from the sarcoplasmic reticulum (SR) by the process of Ca2+-induced Ca2+ release (CICR). The force of heart contraction is thus greatly dependent on the magnitude of Ca2+ release by RyRs; during periods of stress or in the setting of exercise, beta-adrenergic stimulation of ventricular myocytes increases Ca2+ release and thus force of contraction. Beta-adrenergic stimulation increases Ca2+ release by activation of PKA, which phosphorylates phospholamban, thereby relieving its inhibition on the SR Ca 2 pump (SERCA) and increasing SR Ca2+ load. Higher SR Ca2+ load then leads to higher Ca2+ release. Substantial phosphorylation of RyRs is also seen upon beta-adrenergic stimulation, but the role of RyR phosphorylation in normal and diseased states remains controversial. This proposal seeks to establish the physiological impact of RyR phosphorylation, the molecular players involved in this reaction, and its implications for heart failure. Using a combination of phospho-specific antibodies against RyRs, beta-adrenergic stimulation of whole hearts, cellular Ca2+ imaging, and recording of single RyR activity under quasi-physiological conditions, we propose: 1) To identify the protein kinase(s) activated by beta-adrenergic stimulation that phosphorylate RyRs, their specific phosphorylation site(s) in the RyR sequence, and their extent of participation under physiological conditions. 2) To determine the functional output of specific phosphorylation sites in the RyR, by directly recording wild type and mutant RyR channel activity under quasi-physiological conditions. 3) To establish the role of RyR phosphorylation and their counterpart reaction, RyR dephosphorylation, as a pathogenic mechanism for the blunted Ca2+ release characteristic of heart failure. These studies are likely to provide fresh, novel insight into the mechanisms that control RyR phosphorylation and the functional consequences of this process for the Ca2+ homeostasis of cardiac cells. Thus, they may help resolve the controversial role of RyR phosphorylation in the pathogenesis of heart failure.