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: 8740125
• 负责人: CHULHEE KANG
• 金额: $ 35.88万
• 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/8740125
• 关键词: Address; Adult; Affinity; Anabolism; Basic Science; Binding; Buffers; Calcium; Calcium Binding; Calcium Signaling; Calsequestrin; Canada; Cardiac; 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; Solutions; Specific qualifier value; Staging; Stretching; Striated Muscles; Structure; Structure-Activity Relationship; Techniques; Testing; Tissues; Translating; Triad Acrylic Resin; Variant; Ventricular Tachycardia; Work; design; disease phenotype; functional gain; functional loss; functional restoration; human disease; human subject; in vitro testing; in vivo; mouse junctate protein; mutant; operation; polymerization; prevent; research study; skeletal; skills; 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.

细胞内钙信号在横纹肌中达到最大强度和最高频率。 支持它们的是Calequestrin，Casq，以其高钙结合能力，方便的亲和力和两种 体外显示的独特性质：其结合钙的能力明显依赖于游离钙的多少 呈现出钙驱动的聚合趋势。除了这些存储钙的特性外，还有 “门控”功能的证据，通过Casq感知周围的钙浓度，并相应地 诱导钙释放通道，RyR，通过推测由 Triadin，Tr.考虑到Casq中的多个突变，这些功能的相关性变得明显 与严重疾病有关。我们的目标是定义这些独特的特性在体内的运作 体外培养。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，并 率先量化了Casq在成人肌肉中的功能。该计划包括体外和体内试验。 3个假说：(1)细胞内Casq的聚合程度和类型随着[Ca]的耗竭而变化 在蜂窝商店(SR)内。(2)[Ca~(2+)]SR的变化通过Casq和Tr转换为门控变化 RyR。(3)与CPVT(儿茶酚胺能多形性心室)相关的Casq2基因突变 心动过速)，以及M87T，Casq1的一个变异在相当大比例的患者中存在 疾病MH(恶性高热)，通过机制(1)和(2)引起疾病表型。为了测试 (1)我们将在钙缺乏的细胞中检测Casq1的EM结构，测试非聚合的能力 突变Casq1以恢复Casq缺失细胞的功能，并进行同位素钙离子的体外测定 扩散，探索Casq的存在是否改变了Ca的扩散，以及这种影响如何依赖于Casq 聚合反应。对于(2)，我们将探索消除由提供的假定链接对钙信号的影响 在(A)Tr缺失的小鼠和(B)通过表达“诱骗”而急剧失去联系的细胞中的TR，这些寡肽 在Tr序列中绑定Casq。对于(3)，我们将表征11个已知的依赖于钙的物理化学 Casq2的突变体、Casq1的同源突变体及其M87T突变体。然后我们将测试一下 这些突变体可以恢复Casq缺失的小鼠纤维的功能。同时理解这种标志性的生物缓冲区是如何 工作将是主要的回报，长期目标是建立一个可翻译为理性的基础科学基础 应对人类疾病的策略，即：找到致病突变产生 蛋白质在体外鉴定其功能，在体内则说明致病功能的获得或丧失。是一种新的致病基因。 这些阶段的成功完成将使我们在未来的项目迭代中有可能进行设计 救援战略检验了它们的有效性。