Calcins as Membrane-permeable Ligands of Ryanodine Receptors

钙素作为瑞尼定受体的膜渗透性配体

基本信息

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
项目类别：
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

项目摘要

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) 是肌浆网 Ca2+ 释放通道，在可兴奋和不可兴奋细胞的 Ca2+ 信号传导中发挥关键作用。 RyR 之所以得名，是因为它们的特征在很大程度上要归功于兰尼碱，这是一种以高亲和力和特异性与 RyR 结合的植物生物碱。 Ryanodine 一直是 RyRs 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。在寻找可以克服兰尼碱的一些功能和结构缺点的新型配体时，我们在选定的蝎子的毒液中发现了一组肽毒素，称为钙素，对 RyRs 显示出高亲和力和精细的选择性。钙素的决定性特征是它们能够将 RyR 开口稳定在持久的亚导状态。这种作用几乎与兰尼定类似，但与兰尼定不同，钙素快速与 RyR 结合（快速结合速率），从其结合位点自由解离（可逆作用），显示出剂量和序列可变的作用，并且适合衍生化而不会造成受体亲和力的重大损失。 Calcins 还能以惊人的速度和多种效力调节完整心肌细胞中的细胞内 Ca2+，从而作为第一个高动态范围的细胞穿透肽 (CPP) RyR 特异性 Ca2+ 动员剂进入该领域。该研究项目将首先表征并利用这组新型肽毒素，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改参与 RyR 识别和细胞渗透的钙素的结构域，以产生一组功能多样的 CPP，能够以宽动态范围调节 RyR 功能并将货物递送到心肌细胞内部。在第二个目标中，我们将在天然和重组 RyR、完整心肌细胞和 Langendorff 灌注的工作心脏中使用钙素，以产生急性或持续的 RyR 过度活跃期，并揭示 RyR 门控、Ca2+ 触发的心律失常和机电交替的机制。这些研究将使用心室心肌细胞作为表征钙素的细胞模型，但我们的最终目标是为科学界生成一组功能多样的 CPP，它们能够调节 RyR 功能并将货物运送到各种细胞内部。公共健康相关性：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发挥着关键作用，包括收缩、转录调节、酶激活、代谢调节等，但其功能的许多方面仍然不清楚。理解兰尼碱受体的整体作用的一个主要障碍是缺乏特定的配体（选择性地和高亲和力地结合并改变分子活性的分子）。最近，我们发现选定的蝎毒具有一组能够穿透细胞膜并改变兰尼碱受体活性的肽配体。在这项研究中，我们将利用这组新型肽毒素来回答兰尼碱受体结构和功能的一些最基本的问题。因此，此处获得的结果适用于多种细胞。