Roles of Calsequestrin in the Control of Calcium Signals in Health and Disease

Calsequestrin 在控制健康和疾病钙信号中的作用

• 批准号: 9085328
• 负责人: CHULHEE KANG
• 金额: $ 34.22万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9085328
• 关键词: Address; Adult; Affinity; Anabolism; Applied Skills; Basic Science; Binding; Buffers; Calcium; Calcium Binding; Calcium Signaling; Calsequestrin; Canada; Cardiac; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell physiology; Cells; Clinical; Complex; Computer Simulation; Dependence; Deposition; Diagnosis; Diffusion; Disease; Failure; Fiber; Foundations; Frequencies; Functional disorder; Future; Goals; Health; Heart Diseases; Hepatocyte; Homologous Gene; Human; Image; In Vitro; Individual; Ions; Knockout Mice; Lead; Life; Link; Malignant hyperpyrexia due to anesthesia; Measurement; Measures; Mechanics; Mediating; Modification; Molecular; Movement; Mus; Muscle; Mutate; Mutation; Myocardium; Neurons; Null Lymphocytes; Oligopeptides; Outcome; Patients; Permeability; Phenotype; Physical Chemistry; Polymers; Property; Protein Isoforms; Proteins; Proteolysis; Public Health; Recombinants; Resources; Retrieval; Rewards; Role; Signal Transduction; Signaling Molecule; Skeletal Muscle; Specific qualifier value; Staging; Stretching; Striated Muscles; Structure; Structure-Activity Relationship; Techniques; Testing; Tissues; Translating; Triad Acrylic Resin; Variant; Work; design; disease phenotype; functional gain; functional loss; functional restoration; human disease; human subject; in vitro testing; in vivo; mutant; operation; polymerization; prevent; research study; skeletal; stem; triadin; vector

Intracellular Ca signals reach their greatest intensity and highest frequencies in striated muscle. Sustaining them is calsequestrin, Casq, notable for its high Ca-binding capacity, convenient affinity and for two unique properties demonstrated in vitro: a marked dependence of its ability to bind Ca on how much free Ca is present and a Ca-driven tendency to polymerize. In addition to these Ca storing properties, there are evidences for a “gating” function, whereby Casq senses the surrounding Ca concentration and accordingly induces the Ca release channel, RyR, to open or close, through a mechanical link presumably provided by triadin, Tr. The relevance of these functions becomes obvious in view of multiple mutations in Casq that are linked to grave diseases. Our goal is to define the operation in vivo of these unique properties demonstrated in vitro. To this end we joined a lab that has driven the study of Casq's physical chemistry and one that pioneered the quantification of Casq's functions in adult muscle. The plan includes the testing, in vitro and in vivo, of three hypotheses: (1) the extent and type of polymerization of Casq in cells changes as [Ca] depletes inside the cellular store (SR). (2) Changes in [Ca2+]SR are translated, via Casq and Tr, to gating changes in the RyR. (3) Mutations in Casq2 linked to the disease CPVT (catecholaminergic polymorphic ventricular tachycardia), as well as M87T, a variant in Casq1 present in a sizable percentage of patients tested for the disease MH (malignant hyperthermia), cause the disease phenotype through mechanisms (1) and (2). To test (1) we will examine the EM structure of Casq1 in Ca-depleted cells, test the ability of a non-polymerizing mutated Casq1 to restore function in Casq-null cells, and carry out in vitro measurements of isotopic Ca diffusion, probing whether the presence of Casq alters Ca diffusion, and how the effects depends on Casq polymerization. For (2) we will explore the effects on Ca signaling of eliminating the putative link provided by Tr in (a) Tr-null mice and (b) cells acutely deprived of the link by expression of “decoys”, the oligopeptides that bind Casq in the Tr sequence. For (3) we will characterize the Ca-dependent physical chemistry of 11 known mutants of Casq2, the homologous mutants of Casq1 and its M87T variant. We will then test the ability of these mutants to restore function in Casq-null mouse fibers. While understanding how this iconic biobuffer works will be a main reward, the long-term goal is to build a basic science foundation translatable to rational strategies that address human diseases, namely: find the causative mutationgenerate the proteincharacterize its function in vitro  then in vivo specify the pathogenic functional gain or loss. Successful completion of these stages will allow us in future iterations of the project to design possible rescue strategiestest their efficacy.

细胞内Ca信号在横纹肌中达到最大强度和最高频率。 维持它们的是钙螯合蛋白，Casq，以其高钙结合能力，方便的亲和力和两个 在体外证明的独特性质：其结合钙的能力明显依赖于游离钙的多少， 目前和钙驱动的倾向，以减少。除了这些Ca储存特性之外，还有 “门控”功能的证据，Casq借此感知周围的Ca浓度， 诱导钙释放通道RyR打开或关闭，通过一个机械连接，推测是由 triadin，Tr.鉴于Casq中的多个突变，这些功能的相关性变得显而易见， 与严重疾病有关。我们的目标是确定这些独特的性质在体内的运作， 体外为此，我们加入了一个实验室，该实验室推动了对Casq物理化学的研究， 开创了成人肌肉中Casq功能的量化。该计划包括体外和体内试验 体内，三个假设：（1）随着[Ca]耗尽，细胞中Casq聚合的程度和类型发生变化 在蜂窝存储（SR）中。(2)[Ca2+]SR的变化通过Casq和Tr被翻译为细胞内的门控变化。 RyR。(3)Casq2中的突变与CPVT（儿茶酚胺能多形心室肌纤维化）疾病有关。 心动过速），以及M87T，Casq 1的一种变体，在相当大比例的检测了 疾病MH（恶性高热），通过机制（1）和（2）引起疾病表型。测试 (1)我们将在钙耗尽的细胞中检查Casq1的EM结构，测试非聚合Casq1的能力， 突变的Casq 1以恢复Casq缺失细胞的功能，并进行同位素Ca 扩散，探测Casq的存在是否改变Ca扩散，以及影响如何取决于Casq 聚合法对于（2），我们将探索消除由以下提供的假定联系对Ca信号传导的影响： 在（a）Tr缺失小鼠和（B）细胞中，Tr通过表达“诱饵”而被急性剥夺了连接，“诱饵”是 在Tr序列中结合Casq。对于（3），我们将表征11个已知的钙依赖的物理化学 Casq2的突变体、Casq1的同源突变体及其M87T变体。然后我们将测试 这些突变体恢复Casq缺失小鼠纤维中的功能。在了解这个标志性的生物缓冲区 作品将是一个主要的奖励，长期的目标是建立一个基本的科学基础，可翻译的理性 解决人类疾病的战略，即：找到致病突变， 蛋白质组在体外表征其功能，然后在体内确定致病功能的获得或丧失。 这些阶段的成功完成将使我们能够在项目的未来迭代中重新设计 拯救策略的有效性有待检验。