Uncovering the Molecular Basis of Malignant Hyperthermia

描述(由申请人提供)： 我们的总体假设是，RyR1基因突变导致人类恶性高热(MH)和中枢性核心病(CCD)，改变了EC偶联大分子复合体的稳定性和钙信号的动态，这些变化导致对触发MH的药物的易感性，并造成与CD相关的弱点。来自我们的基因异常(MDG)和速度障碍小鼠模型的数据已经确定了负责这两种蛋白之间双向信号传递的表膜慢电压门控钙通道(y is-DHPR)和肌浆网(SR)RyR1的关键区域。我们最近还展示了RyR1、Vis-DHPR和细胞膜上的钙离子进入通道之间的三向相互作用。MH突变的RyRs和Y IS-DHPR的体外研究表明，它们的表型与MH易感的人和猪的肌肉相似。已经建立了三种表达RyR1的小鼠模型，该模型带有MH/CD突变。将用其他RyR1突变创建更多的小鼠，以使我们能够研究表型严重程度和突变位置之间的关系。项目1-4的主题密切相关，每个项目PI的专业知识都是独一无二的，对拟议项目的跨学科目标至关重要。行政、组织培养转基因动物、人体组织和形态核心将为四个项目调查人员提供通用试剂和形态研究的统一资源。研究范围包括MH和CD小鼠的建立和广泛的表型(项目1和3)、分离肌肉三联体中蛋白质功能和相互作用的研究(项目2)、突变对RyR1结构和生化的影响(项目3)以及它们对肌肉和树突状细胞的细胞生理学的影响(项目1、2和4)。这项计划项目提出的工作的预期结果是更好地理解RyR1的MH和CD突变如何以不同的方式扰乱静息和活动期间细胞质钙的动态，它们如何促进微妙的到灾难性的细胞功能障碍，以及它们与遗传背景的关系。

DESCRIPTION (provided by applicant): Our overall hypothesis is that the mutations in RyR1 responsible for human Malignant Hyperthermia (MH) and Central Core Disease (CCD) alter the stability of the EC coupling macromolecular complex and the dynamics of Ca2+ signaling, These changes are responsible for susceptibility to agents that trigger MH and to create the weakness associated with CCD. Data from our dysgenic (mdg) and dyspedic mouse models have defined the critical regions of the slow voltage gated Ca2+ channel in the surface membrane (y is-DHPR) and RyR1 of the sarcoplasmic reticulum (SR) that are responsible for bi-directional signaling between these two proteins. We have also recently demonstrated a three-way interaction between RyR1, the vis-DHPR and a Ca2+-entry channel in the surface membrane. In vitro studies of RyRs and Y is-DHPRs with MH mutations expressed in null cell lines have demonstrated that they have a phenotype similar to muscle from MH susceptible humans and pigs. Three mouse models have been created expressing RyR1 with MH/CCD nutations. Additional mice will be created with other RyR1 mutations to allow us to study the relationship between severity of the phenotype and the site of a mutation. The themes of Projects 1-4 are closely interrelated, and the expertise from each of the project PI's is unique and essential to the interdisciplinary goals of the proposed program. The administrative, tissue culture transgenic animal, human tissue and morphology cores will provide a unifying resource for common reagents and morphology studies for the four project investigators. The scope of investigations will range from creation and extensive phenotyping of MH and CCD mice (Projects 1 and 3), studies of protein function and interactions in isolated muscle triads (Project 2), influence of mutations on the RyR1 structure and biochemistry (Project 3), and their influence on the cellular physiology of muscle and dendritic cells (Projects 1, 2, & 4). The expected outcome of the work proposed by this Program Project is a better understanding how MH and CCD mutations of RyR1 differentially perturb the dynamics of cytoplasmic Ca2+ at rest and during activity, how they promote subtle to catastrophic cellular dysfunctions and their relationship to genetic background.