Natural Agonists of Ryanodine Receptors: Structure-function Relationship and Antiarrhythmic Properties

摘要 Ryanodine受体(RyR)是肌浆网钙释放通道，在肌浆网钙离子释放中起重要作用。 可兴奋和不可兴奋细胞的钙信号转导。RYR之所以得名，是因为它们的特点是 这在很大程度上要归功于ryanodine，一种以高亲和力和特异性与RyRs结合的植物生物碱。兰诺定有 作为RyRs的无价配体，但其功能作用复杂，阻碍了其在细胞研究中的应用。在……里面 寻找新的配体可以克服兰诺定的一些功能和结构上的缺点，我们 在选定的蝎子的毒液中发现了一组多肽配体，称为钙蛋白，显示出高亲和力和 对RYR的精致选择性。钙蛋白的决定性特征是其稳定RyR开口的能力 一种持久的次导电态。这种作用几乎与ryanodine类似，但与ryanodine不同的是，钙蛋白 快速结合RyRs(快速结合率)，自由解离其结合部位(可逆效应)，表现出剂量- 和序列可变效应，并且易于衍生化，而不会在受体亲和力方面遭受重大损失。 Calcins还以惊人的速度和不同程度地调节完整心肌细胞内的钙离子。 效价高，从而作为第一个细胞穿透肽(CPP)RyR特异性高钙激动剂进入该领域 动态范围。这项研究计划将首先表征这组新的多肽配体，然后开发 在分子、细胞和整个心脏水平上揭示RyR功能的基本机制。我们的多学科 计划有明确的交付成果和里程碑，可能包含两个具体目标。在第一个目标中，我们 将首先识别和修饰参与RyR识别和细胞穿透的钙蛋白的结构域，以 产生一组功能不同的CPP，能够以宽动态范围调制RyR函数，并且 将货物运送到心肌细胞内部。在第二个目标中，我们将在完整的 心肌细胞，兰登多夫灌流的工作心脏和完整的动物，以创造急性或持续性的RyR 并揭示RyR门控、肌质网钙负荷和钙离子触发的心律失常的机制。这些研究使用 儿茶酚胺能多形性室性心动过速动物模型研究 钙依赖型心律失常；结果可能适用于其他心肌病，在这些心肌病中控制负荷 肌浆钙离子可能是可取的。

ABSTRACT Ryanodine receptors (RyR) are sarcoplasmic reticulum Ca2+ release channels that play a critical role in Ca2+ signaling of excitable and non-excitable cells. RyRs owe their name to the fact that they were characterized in great part thanks to ryanodine, a plant alkaloid that binds to RyRs with high affinity and specificity. Ryanodine has been an invaluable ligand of RyRs, but its functional effects are complex and hamper its use in cellular studies. In search of novel ligands that could overcome some of the functional and structural disadvantages of ryanodine, we found in the venom of selected scorpions a set of peptide ligands, termed calcins, displaying high affinity and exquisite selectivity against RyRs. The defining characteristic of calcins is their capacity to stabilize RyR openings in a long-lasting subconducting state. This effect is nearly analogous to that of ryanodine, but unlike ryanodine, calcins bind rapidly to RyRs (fast association rate), freely dissociate from their binding site (reversible effect), display a dose- and sequence-variable effect, and are amenable for derivatization without undergoing major loss in receptor affinity. Calcins also modulate intracellular Ca2+ in intact cardiomyocytes with remarkable speed and with several degrees of potency, thus entering the field as the first cell-penetrating peptides (CPP) RyR-specific Ca2+ mobilizer of high dynamic range. This research program will characterize first and then exploit this novel group of peptide ligands to unravel fundamental mechanisms of RyR function at the molecular, cellular and whole heart level. Our multidisciplinary program, with well-defined deliverables and milestones, may be enveloped in two specific aims. In the first aim, we will first identify and modify the structural domains of calcins involved in RyR recognition and cell penetration to generate a group of functionally diverse CPPs capable of modulating RyR function with wide dynamic range and of delivering cargo to the interior of cardiomyocytes. In the second aim, we will use native and mutant calcins on intact cardiomyocytes, Langendorff-perfused working hearts and intact animals to create acute or sustained periods of RyR hyperactivity and reveal mechanisms of RyR gating, SR Ca2+ load and Ca2+-triggered arrhythmias. These studies use animal models of catecholaminergic polymorphic ventricular tachycardia to develop a novel paradigm for the treatment of calcium-dependent arrhythmias; results may be applied to other cardiomyopathies where controlled unloading of SR Ca2+ may be desirable.