AKAP-dependent regulation of Cardiac SR Ca handling

项目摘要/摘要 β-肾上腺素能受体(β-AR)激活影响心脏兴奋收缩偶联 通过SERCA和SERCA协同增加肌浆网(SR)钙摄取 通过Ryanodine受体(RyR)通道释放。肌浆网钙摄取减少和肌浆网钙敏化 释放有助于降低心力衰竭(HF)的肌浆网钙含量，是关键的治疗方法 高频下的目标。然而，在我们对？AR的基本理解中，存在着关键的差距 在心肌细胞中调节这些肌质网钙的处理过程，我们的团队很好地- 准备发表演讲。磷脂蛋白(PLB)基本抑制了SR对钙的摄取，这种作用是 PKA使PLB磷酸化而缓解。一个量化的机制问题是，一个小的 细胞中的PKA分子的数量可以迅速磷酸化更多的(&gt；200- 褶皱)和广泛分布的PLB，以均匀地加速[Ca]i下降和SR Ca负荷 在？-AR激活时。不太可能将PKA专门锚定在每一小簇PLB附近 从定量的角度来看，分子就足够了。新数据表明，公共汽车 磷酸化可以降低A-激酶锚定蛋白AKAP7的PLB亲和力。我们将测试 一种假说是，当？AR被激活时，与非磷酸化原球蛋白结合的AKAP7 使附近的PLB分子磷酸化，但随后分离，只是再次快速结合到 未磷酸化的原核生物的邻近区域(也就是沿着SR“悬停”，正在磷酸化 顺序的PLB簇)。这一新的范式不同于标准，接受了1：1 其他AKAP和他们的靶子之间的关系，可能是快速肾上腺素能战斗的关键-否则- 飞行反应。AIM 1将测试AKAP7移动性如何受PLB结合和 磷酸化以加速肾上腺素能心脏震颤。目标2将测试以下各项的后果 干扰AKAP7-PLB相互作用对SERCA时空传播的影响 单个肌细胞的激活。AIM 3将测试AKAP7-CaMKII相互作用及其潜力 对PLB和RyR的影响。这项工作将提供新的和明确的机制数据关于 心肌细胞内肾上腺素能快速同步激活的基本机制 还有心。这可能会提供一种新型的AKAP功能，其中靶向转位的 AKAP将信号放大到非常多的目标站点。

Project Summary/ Abstract Beta-adrenergic receptor (β-AR) activation affects cardiac excitation-contraction coupling (ECC) through coordinated increases in sarcoplasmic reticulum (SR) Ca uptake by SERCA and release via ryanodine receptor (RyR) channels. Reduced SR Ca uptake and sensitized SR Ca release contribute to reduced SR Ca content in heart failure (HF), and are key therapeutic targets in HF. However, there are key gaps in our fundamental understanding of ß-AR regulation of these SR Ca handling processes in cardiac myocytes, which our team is well- poised to address. SR Ca uptake is basally inhibited by phospholamban (PLB), an effect that is relieved by PLB phosphorylation by PKA. A quantitative mechanistic question is how a small number of PKA molecules in the cell can rapidly phosphorylate the far more numerous (>200- fold) and widely distributed PLB, to uniformly accelerate [Ca]i decline and SR Ca load upon ß-AR activation. It is unlikely that dedicated anchoring of PKA near each small cluster of PLB molecules would suffice from a quantitative standpoint. New data suggest that PLB phosphorylation can decrease PLB affinity of A-kinase anchoring protein, AKAP7. We will test the hypothesis that upon ß-AR activation, the AKAP7 that is bound to un-phosphorylated PLB phosphorylates nearby PLB molecules, but then detaches, only to bind again quickly to a neighboring area of unphosphorylated PLBs (i.e. “hovering” along the SR, phosphorylating sequential PLB clusters). This new paradigm, which differs from the standard, accepted 1:1 relationship between other AKAPs and their targets, may be critical to rapid adrenergic fight-or- flight response. Aim 1 will test how AKAP7 mobility is influenced by PLB binding and phosphorylation to expedite adrenergic cardiac lusitropy. Aim 2 will test consequences of disrupting the AKAP7-PLB interaction with respect to spatiotemporal spread of SERCA activation in single myocytes. Aim 3 will test for AKAP7-CaMKII interaction and its potential effects on PLB & RyR. This work will provide novel and clear mechanistic data regarding the fundamental mechanism of rapid synchronous adrenergic activation throughout the myocyte and heart. This may present a novel type of AKAP function, where targeted translocation of the AKAP amplifies signaling to very large numbers of target sites.