beta-adrenergic modulation of cardiac ryanodine receptor

描述（由申请人提供）：在心脏中，通过L型Ca 2+通道（DHPR）的少量Ca 2+内流触发兰尼碱受体（RyR）的开放，进而通过Ca 2+诱导的Ca 2+释放（CICR）过程从肌浆网（SR）释放大量Ca 2+。因此，心脏收缩力在很大程度上取决于RyR释放的Ca 2+的大小;在应激期间或在运动环境中，心室肌细胞的β-肾上腺素能刺激增加Ca 2+释放，从而增加收缩力。 β-肾上腺素能刺激通过激活PKA增加Ca 2+释放，PKA使受磷蛋白磷酸化，从而缓解其对SR Ca 2泵（SERCA）的抑制并增加SR Ca 2+负荷。更高的SR Ca 2+负荷则导致更高的Ca 2+释放。在β-肾上腺素能刺激后也观察到RyR的大量磷酸化，但RyR磷酸化在正常和疾病状态中的作用仍然存在争议。该提案旨在确定RyR磷酸化的生理影响，参与该反应的分子参与者及其对心力衰竭的影响。利用磷酸化特异性抗RyR抗体、β-肾上腺素能刺激全心脏、细胞Ca ~（2+）成像和在准生理条件下记录单个RyR活性的组合，我们提出：1）鉴定由β-肾上腺素能刺激激活的使RyR磷酸化的蛋白激酶、它们在RyR序列中的特异性磷酸化位点以及它们在生理条件下的参与程度。2)通过在准生理条件下直接记录野生型和突变型RyR通道活性，确定RyR中特定磷酸化位点的功能输出。3)确定RyR磷酸化及其对应反应RyR去磷酸化的作用，作为心力衰竭的钝性Ca 2+释放特征的致病机制。这些研究很可能提供新鲜的，新颖的洞察力的机制，控制RyR磷酸化和功能的后果，这一过程中的Ca 2+稳态的心脏细胞。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.