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

描述(由申请人提供)：此提案的PI是布里格姆妇女医院/哈佛医学院的麻醉学讲师。这项提案中概述的工作旨在资助肌肉生理学的两个领域的指导培训，以发展两项尖端技术的新技能。这种有指导的培训将帮助PI发展新的工作协作，导致申请R01级别的资金，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员。这项提案的导师是G.Stahl博士，他是一位在Biacore分析方面拥有丰富经验的独立科学家；K.Beam博士，一位在电生理学方面拥有丰富经验的独立科学家，他是一名放弃肌肉生理学领域的研究员；J.Lopez博士，一位在肌肉细胞微电极记录方面拥有丰富经验的独立调查人员。这项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在最高水平进行生物医学研究。这项研究的长期目标是定位骨骼兰尼定受体(RyR1)和DHPR(1S和1a亚单位)一级序列中的结构决定子(S)，参与这两个通道在骨骼肌收缩过程中的直接相互作用，并评估这些相互作用在体内对RyR/DHPR信号的功能影响。假设：(1s和(1a)DHPR亚基通过复杂的物理相互作用与RyR1相连，这需要两个受体中的多个相互作用点。参与这些相互作用的RyR1结构域(S)是RyR1所特有的，因为来自非偶联受体(即RyR3)的同源区域(S)不支持这种相互作用。具体目的1.研究DHPR oc1s和(5ia)亚基与wt-RyR1和RyR3体外相互作用的能力。纯化的RyRs将被固定在能够支持脂质双层的Biacore传感器芯片中，并将通过在活化的表面上注入全长A1或P1a亚单位来监测与DHPR的相互作用。具体目的2.确定RyR1与DHPR相互作用的特定结构域。我们将研究(1S和(1a)与RyR1/RyR3嵌合受体文库的结合特性，该文库包含几个区域RyR1到RyR3背景中。具体目的3.确定DHPR(1s和(1a)参与与RyR1的物理相互作用的最小本质结构域。我们将检测嵌合体(1S/(1c)亚基在体外与纯化的RyRs相互作用的能力。具体目的4.研究(1s/RyR1和(1a/RyR1)相互作用对EC偶联、逆行信号转导和静息钙离子的作用。广泛的RyR3/RyR1和DHPR嵌合文库(1S/(1c结构将在单和双敲除肌管中表达(RyR、DHPR和RyR/DHPR为空))，并使用钙成像、膜片钳和钙微电极进行测试。相关性：充分了解骨骼肌收缩过程中发生的复杂信号传递过程，对于开发旨在改善病理生理状态下的收缩能力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.