Structural/functional interaction between RyR1 and DHPR alpha1s and Beta1a isofor

描述（由申请人提供）：本提案的PI是布里格姆妇女医院/哈佛医学院的麻醉讲师。本提案中概述的工作旨在资助肌肉生理学两个领域的指导培训，以开发两种尖端技术的新技能。该指导培训将帮助PI发展新的工作合作，从而申请R 01级资金，并帮助PI从目前的职位过渡到哈佛医学院的独立学术研究者。本书的导师是G博士。斯塔尔，他是一位独立的科学家，在BIACORE分析方面有着丰富的经验; Beam博士是一位在电生理学方面拥有丰富经验的独立科学家，他是肌肉生理学领域的研究者，而J.洛佩斯博士是一位在肌肉细胞微电极记录方面拥有丰富经验的独立研究者。科学工作将在哈佛医学院和UCHSC进行，这两个世界级的科学机构进行最高水平的生物医学研究。本研究的长期目标是定位骨骼肌Ryanodine受体（RyR 1）和DHPR（1 s和1a）亚基一级序列中参与骨骼肌收缩过程中这两个通道直接相互作用的结构决定子，并评估这些相互作用对体内RyR/DHPR信号传导的功能影响。假设：β 1 s和β 1a DHPR亚基通过一种复杂的物理相互作用与RyR 1相连，这种相互作用需要两种受体中的多个相互作用点。参与这些相互作用的RyR 1的结构域是RyR 1特异性的，因为来自非偶联受体（即RyR 3）的同源区域不支持这种相互作用。具体目标1。研究DHPR α 1 s和β 5ia亚基在体外与野生型RyR 1和RyR 3相互作用的能力。将纯化的RyR固定在能够支持脂质双层的BIACORE传感器芯片中，并通过在活化表面上注射全长α 1 s或β 1 a亚基来监测与DHPR的相互作用。具体目标2。确定RyR 1与DHPR相互作用的特异性结构域。我们将研究β 1 s和β 1a与嵌合RyR 1/RyR 3受体库的结合特性，所述嵌合RyR 1/RyR 3受体库包含RyR 1到RyR 3背景中的几个区域。具体目标3。目的：确定与RyR 1相互作用的DHPR（1 s）和（1a）的最小必需结构域。我们将检测嵌合的β 1 s/β 1c亚基在体外与纯化的RyR相互作用的能力。具体目标4。研究β 1 s/RyR 1和β 1a/RyR 1相互作用对EC偶联、逆行信号和静息Ca ~（2+）的作用。嵌合RyR 3/RyR 1和DHPR（1 s/1c）构建体的广泛文库将在单和双敲除肌管（RyR、DHPR和RyR/DHPR为空）中表达，并使用钙成像、膜片钳和钙微电极进行测试。相关性：全面了解骨骼肌收缩过程中发生的精细信号传导过程，对于开发旨在改善病理生理状态下收缩性的新疗法具有重要意义。

DESCRIPTION (provided by applicant): The PI of this proposal is an Instructor in Anesthesia at Brigham and Women's Hospital/Harvard Medical School. The work outlined in this proposal is intended to fund mentored training in two areas of muscle physiology to develop new skills in two cutting edge technologies. This mentored training will help the PI in the development of new working collaboration, leading to an application of R01 level funding and help in the transition of the PI from his current position to an independent academic investigator at Harvard Medical School. The mentors in this proposal are Dr. G. Stahl, who is an independent scientist with extensive experience in BIACORE analysis; Dr. K. Beam, an independent scientist with extensive experience in electrophysiology who is a renounce investigator in the field of muscle physiology and Dr. J. Lopez, an independent investigators with vast experience in microelectrode recording in muscle cells. The scientific work will be carried out at Harvard Medical School and UCHSC, two world-class scientific institutions where biomedical research is performed at the highest level. The long-term goal of the proposed study is to localize the structural determinant(s), within the primary sequence of skeletal ryanodine receptor (RyR1) and DHPR (1s and (1a subunit, involved in the direct interaction that these two channels engage on during skeletal muscle contraction and assess the functional effect of these interactions on RyR/DHPR signaling in vivo. Hypothesis: (1s and (1a DHPR subunits are linked to RyR1 through a complex physical interaction that requires multiple interacting points in both receptors. The domain(s) of RyR1 involved in these interactions are specific of RyR1 since homologous region(s) from non-coupled receptors (i.e. RyR3) do not support such interaction. Specific Aim 1. To study the ability of DHPR oc1s and (5ia subunits to interact in vitro with wt-RyR1 and RyR3. Purified RyRs will be immobilized in BIACORE sensor chips capable of supporting lipid bilayer and interactions with DHPRs will be monitored by injecting either full-length a1s or p1a subunit over the activated surface. Specific Aim 2. To define specific domains of RyR1 involved in the interaction with DHPR. We will study the binding properties of (1s and (1a to a library of chimeric RyR1/RyR3 receptors containing several regions RyR1 into a RyR3 background. Specific Aim 3. To define the minimal essential domain of DHPR (1s and (1a involved in the physical interaction with RyR1. We will assay the ability of chimeric (1s/(1c subunits to interact in vitro with purified RyRs. Specific Aim 4. To study the functional role of the identified (1s/RyR1 and (1a/RyR1 interactions on EC coupling and retrograde signaling and resting Ca2+. An extensive library of chimeric RyR3/RyR1 and DHPR (1s/(1c constructs will be expressed in single and double knockout myotubes (nulls for RyRs, DHPRs and RyR/DHPR) and tested using calcium imaging, patch-clamp and calcium microelectrodes. Relevance: A full understanding of the elaborate signaling processes that take place during skeletal muscle contraction has important implications for the development of new therapies aimed at improving contractility on pathophysiologic states.