Calcins as Membrane-permeable Ligands of Ryanodine Receptors

钙素作为瑞尼定受体的膜渗透性配体

• 批准号: 8098484
• 负责人: Hector H Valdivia
• 金额: $ 35.57万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8098484
• 关键词: Active Sites; Acute; Affinity; Agonist; Amino Acids; Arrhythmia; Binding; Binding Sites; Calcium Channel; Cardiac Myocytes; Cell model; 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; Kinetics; Left; Ligands; Location; Malignant hyperpyrexia due to anesthesia; Maps; Membrane; Metabolic; Molecular; Muscle Cells; Myocardium; Names; Paper; Penetration; Peptides; Plant alkaloid; Play; Proteins; Recombinants; Regulation; Research; Role; RyR1; RyR2; Ryanodine; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Scorpion Venoms; Scorpions; Signal Transduction; Skeletal Muscle; Specificity; Speed; Structure; Syndrome; Testing; Toxin; Transcriptional Regulation; Venoms; Ventricular; Work; dipole moment; imperatoxin A; multidisciplinary; mutant; novel; programs; receptor; success

DESCRIPTION (provided by applicant): 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 toxins, 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 toxins 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 calcins on native and recombinant RyR, in intact cardiomyocytes, and Langendorff-perfused working hearts to create acute or sustained periods of RyR hyperactivity and reveal mechanisms of RyR gating, Ca2+-triggered arrhythmias and electromechanical alternans. These studies will use ventricular cardiomyocytes as the cell model for characterization of calcins, but our ultimate goal is to generate for the scientific community a group of functionally diverse CPPs capable of modulating RyR function and of carrying cargo to the interior of a wide range of cells. PUBLIC HEALTH RELEVANCE: Ryanodine receptors are ubiquotous intracellular calcium channels that play a critical role in many aspects of cell function, including contraction, transcriptional regulation, enzyme activation, metabolic regulation, etc. but many aspects of their function still remain obscure. A major obstacle towards understanding the integral role of ryanodine receptors is the absence of specific ligands (molecules that bind and alter the activity of a molecule selectively and with high affinity). Recently, we discovered that selected scorpion venoms possess a group of peptide ligands that are able to penetrate cellular membranes and alter ryanodine receptor activity. In this study, we will exploit this novel group of peptide toxins to answer some of the most fundamental questions of ryanodine receptor structure and function. Results obtained here are therefore applicable to a wide range of cells.

描述（由申请人提供）：ryanodine受体（RYR）是肌质网Ca2+释放通道，它们在可激发和不可驱行的细胞的Ca2+信号传导中起关键作用。里尔斯（Ryrs）的名字归功于他们在很大程度上归功于Ryanodine的特征，这是一种与高亲和力和特异性的Ryrs结合的植物生物碱。 ryanodine一直是Ryrs的宝贵配体，但其功能作用是复杂的，并阻碍了其在细胞研究中的使用。为了寻找可以克服ryanodine的某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毒素，称为钙蛋白，称为高亲和力，对Ryrs表现出高亲和力和精致的选择性。 Calcins的定义特征是它们以持久的子导状态稳定RYR开口的能力。这种作用几乎与瑞氨烷类似，但与瑞氨烷不同，Carcins与RYRS（快速关联速率）迅速结合，与其结合位点自由解离（可逆效应），表现出剂量和序列可变效果，并且在受体亲身亲身亲身亲身亲身亲身亲身亲属的情况下表现出可取代的剂量和序列可变效应。钙蛋白还以显着速度和几个效力的完整心肌细胞中调节细胞内Ca2+，从而作为第一个细胞穿透性肽（CPP）Ryr特异性CA2+高动态范围的动员进入该场。该研究计划将首先表征，然后利用这种新型的肽毒素群体在分子，细胞和整个心脏水平上揭示RYR功能的基本机制。我们的多学科计划具有明确的可交付成果和里程碑，可以包裹在两个特定的目标中。在第一个目标中，我们将首先识别并修改参与RYR识别和细胞渗透的钙蛋白的结构结构域，以生成一组功能多样的CPP，能够调节具有广泛动态范围的RYR功能并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和Langendorff垂直的工作心脏上使用钙蛋白，以创建RYR多功能的急性或持续时间，并揭示Ryr Gating的急性或持续时间，并揭示Ryr Gating的机制，Ca2+触发的ARRHILTHMIAS和电子力学替代品。这些研究将使用心室心肌细胞作为钙蛋白表征的细胞模型，但是我们的最终目标是为科学界生成一组功能多样的CPP，能够调节RYR功能并将货物载运到广泛的细胞内部。 公共卫生相关性：ryanodine受体是普遍存在的细胞内钙通道，在细胞功能的许多方面起着至关重要的作用，包括收缩，转录调控，酶激活，代谢调节等，但其功能的许多方面仍然保持晦涩难懂。理解ryanodine受体不可或缺的作用的主要障碍是缺乏特定的配体（分子有选择地和高亲和力结合和改变分子的活性）。最近，我们发现选定的蝎子毒液具有一组能够穿透细胞膜并改变ryanodine受体活性的肽配体。在这项研究中，我们将利用这一新型的肽毒素组来回答瑞氨烷受体结构和功能最基本的问题。因此，此处获得的结果适用于广泛的细胞。