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

兰尼定受体的天然激动剂：结构-功能关系和抗心律失常特性

• 批准号: 9650244
• 负责人: Hector H Valdivia
• 金额: $ 46.18万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9650244
• 关键词: Active Sites; Acute; Adrenergic alpha-Antagonists; Affinity; Agonist; Amino Acids; Animal Model; Animals; Arrhythmia; Binding; Binding Sites; Calcium; Calcium Channel; Cardiac Myocytes; Cardiomyopathies; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell physiology; Cells; Cellular Membrane; Central Core Myopathy; Characteristics; Charge; Communities; Complex; Disadvantaged; Dissociation; Dose; Enzyme Activation; Functional disorder; Generations; Goals; Heart; Heart failure; Hyperactive behavior; Ions; Kinetics; Ligands; Location; Malignant hyperpyrexia due to anesthesia; Maps; Metabolic; Molecular; Muscle Cells; Myocardium; Names; Penetration; Peptides; Pharmacology; Plant alkaloid; Play; Property; Proteins; Receptor Activation; Recombinants; Regulation; Research; Role; RyR2; Ryanodine; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Scorpion Venoms; Scorpions; Signal Transduction; Skeletal Muscle; Specificity; Speed; Structure-Activity Relationship; Syndrome; Testing; Transcriptional Regulation; Venoms; Ventricular; dipole moment; experimental study; functional group; imperatoxin A; multidisciplinary; mutant; novel; prevent; programs; receptor; receptor function; receptor structure function; success; thermostability

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.

抽象的 Ryanodine 受体 (RyR) 是肌浆网 Ca2+ 释放通道，在 可兴奋和不可兴奋细胞的 Ca2+ 信号传导。 RyR 之所以得名，是因为它们的特征在于 这很大程度上归功于兰尼碱，一种植物生物碱，能够以高亲和力和特异性与 RyR 结合。瑞安丁有 一直是 RyR 的宝贵配体，但其功能作用很复杂，阻碍了其在细胞研究中的应用。在 为了寻找可以克服兰尼定的一些功能和结构缺点的新型配体，我们 在选定的蝎子的毒液中发现了一组肽配体，称为钙素，具有高亲和力和 对 RyRs 具有出色的选择性。钙素的决定性特征是其稳定 RyR 开口的能力 持久的亚导状态。这种作用几乎与兰尼定相似，但与兰尼定不同的是，钙素 快速与 RyR 结合（快速结合速率），从其结合位点自由解离（可逆效应），显示剂量- 和序列可变效应，并且易于衍生化而不会造成受体亲和力的重大损失。 钙素还可以以惊人的速度和几个程度调节完整心肌细胞中的细胞内 Ca2+ 效力，从而作为第一个细胞穿透肽 (CPP) RyR 特异性 Ca2+ 动员剂进入该领域 动态范围。该研究计划将首先表征然后利用这组新型肽配体来 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 具有明确定义的可交付成果和里程碑的计划可能包含两个具体目标。在第一个目标中，我们 首先将识别并修改参与 RyR 识别和细胞渗透的钙素的结构域 生成一组功能多样的 CPP，能够以宽动态范围调节 RyR 功能，并且 将货物运送到心肌细胞内部。在第二个目标中，我们将在完整的钙素上使用天然和突变的钙素 心肌细胞、Langendorff 灌注的工作心脏和完整的动物以产生急性或持续的 RyR 期 机能亢进并揭示 RyR 门控、SR Ca2+ 负荷和 Ca2+ 触发心律失常的机制。这些研究使用 儿茶酚胺能多形性室性心动过速动物模型，以开发新的治疗范例 钙依赖性心律失常；结果可能适用于其他心肌病，其中控制卸载 SR Ca2+ 可能是理想的。