Sub-Project #4

恶性高热（MH）和中央核心病（CCD）是由骨骼肌突变引起的 兰尼碱受体（RyR 1）。虽然RyR 1中的MH和CCD突变改变了RyR 1与RyR 2之间的机械偶联， 肌膜二氢吡啶受体（DHPR）和肌浆钙释放通道 网（SR），这些突变对多种亚细胞Ca 2+转运过程的综合影响是 不太了解。该项目的长期目标是通过以下方法确定细胞/分子机制： RyR 1中的哪个MH和CCD突变改变了肌膜、SR和 线粒体（“Ca 2+信号三联体”）。具体而言，本项目将检验“MH/CCD”假设， RyR 1的突变增强了兴奋偶联的Ca 2+进入（ECCE）活性，使电压和配体门控的 SR Ca ~（2+）释放，并改变EC偶联过程中线粒体Ca ~（2+）摄取。“目标1将 表征几种常见RyR 1 MH/CCD突变对DHPR-RyR 1双向偶联的影响， 骨骼肌管和完全分化的肌纤维来源于由Core B产生的MH基因敲入小鼠。 目的#2将测试RyR 1中的MH/CCD突变是否通过促进Ca 2+的消耗来升高稳态静息Ca 2+。 SR Ca ~（2+）和增加肌膜ECCE通道活性。实验将使用SR靶向，Ca 2 +- 敏感的荧光“cameleon”，直接报告SR Ca 2+和全细胞膜片钳的变化 监测幼儿保育和教育活动的变化。目标#3将确定线粒体 三联体靶向和局部SR-线粒体Ca 2+信号传导被RyR 1中的MH/CCD突变改变。 与核心D合作的实验将使用电子显微镜来评估线粒体形态， 定位和三联体靶向正常和MH/CCD敲入小鼠的FOB纤维。功能实验 将使用共聚焦显微镜，高速Ca 2+成像和神经靶向比率pericam， 报告了MH突变对线粒体Ca 2+的幅度、动力学和电压依赖性的影响。 EC偶联期间的变化。此外，将进行与目标1-3中所述的那些平行的实验。 在由对照个体和MHS患者的肌肉样品产生的人肌管中进行 在RyR 1中具有与用于制备敲入小鼠的那些类似的突变（例如R163 C和G2435 R）。为 在这些实验中，由核心C从对照个体和已知免疫缺陷的患者中收集的人类肌肉样品， 核心B将使用基因型和IVCT结果来繁殖生成所需的人成肌细胞。 项目4中的肌管培养物。这个项目将联合收割机结合分子生物学，小鼠遗传学， 电生理学，共聚焦/电子显微镜，和高速Ca 2+成像，以评估机制， 该MH/CCD突变改变了Ca 2+信号三联体的功能。

Malignant hyperthermia (MH) and central core disease (CCD) arise from mutations in the skeletal muscle ryanodine receptor (RyR1). Although MH and CCD mutations in RyR1 alter mechanical coupling between sarcolemmal dihydropyridine receptors (DHPRs) and opposing Ca2+ release channels of the sarcoplasmic reticulum (SR), the integrated effects of these mutations on multiple subcellular Ca2+ transport processes are poorly understood. The long-term goal of this project is to determine the cellular/molecular mechanisms by which MH and CCD mutations in RyR1 alter Ca2+ signaling interactions between the sarcolemma, SR, and mitochondria (the "Ca2+ signaling triad"). Specifically, this project will test the hypothesis that "MH/CCD mutations in RyR1 enhance excitation coupled Ca2+ entry (ECCE) activity, sensitize voltage- & ligand-gated SR Ca2+ release, and alter mitochondrial Ca2+ uptake during EC coupling." Aim #1 will characterize effects of several common RyR1 MH/CCD mutations on bi-directional DHPR-RyR1 coupling in skeletal myotubes and fully differentiated muscle fibers derived from MH knock-in mice generated by Core B. Aim #2 will test if MH/CCD mutations in RyR1 elevate steady-state resting Ca2+ by promoting a depletion of SR Ca2+ and increasing the activity of sarcolemmal ECCE channels. Experiments will use SR-targeted, Ca2+- sensitive fluorescent "cameleons" to directly report changes in SR Ca2+ and whole-cell patch clamp measurements to monitor changes in ECCE activity. Aim #3 will determine the degree to which mitochondrial triad targeting and local SR-mitochondrial Ca2+ signaling is altered by MH/CCD mutations in RyR1. Experiments in collaboration with Core D will use electron microscopy to assess mitochondrial morphology, localization, and triad targeting in FOB fibers of normal and MH/CCD knock-in mice. Functional experiments will use confocal microscopy, high-speed Ca2+ imaging and mitochondrial-targeted ratiometric pericam to report effects of MH mutations on the magnitude, kinetics, and voltage-dependence of mitochondrial Ca2+ changes during EC coupling. Additionally, parallel experiments to those described in Aims 1-3 will be conducted in human myotubes generated from muscle samples of control individuals and MHS patients harboring analogous mutations in RyR1 (e.g. R163C and G2435R) to those used to make knock-in mice. For these experiments, human muscle samples collected by Core C from control individuals and patients of known genotypes and IVCT results will be used by Core B to propagate human myoblasts required for generating myotube cultures in Project 4. This project will combine the tools of molecular biology, mouse genetics, electrophysiology, confocal/electron microscopy, and high-speed Ca2+ imaging to asses the mechanisms by which MH/CCD mutations alter the function with the Ca2+ signaling triad.