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

描述（由申请人提供）：本提案的 PI 是布莱根妇女医院/哈佛医学院的麻醉讲师。该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。这种指导培训将帮助 PI 发展新的工作合作，从而申请 R01 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员。本提案的导师是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 博士是一位在电生理学方面拥有丰富经验的独立科学家，也是肌肉生理学领域的一名放弃研究人员；J. Lopez 博士是一位独立研究人员，在肌肉细胞微电极记录方面拥有丰富的经验。科学研究工作将在哈佛医学院和 UHSC 进行，这两个世界一流的科研机构在生物医学研究方面处于最高水平。拟议研究的长期目标是定位骨骼兰尼碱受体 (RyR1) 和 DHPR (1s 和 (1a 亚基) 的一级序列中的结构决定因素，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对体内 RyR/DHPR 信号传导的功能影响。假设：(1s 和 (1a DHPR 亚基与RyR1 通过复杂的物理相互作用，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 结构域是 RyR1 特有的，因为非偶联受体（即 RyR3）的同源区域不支持这种相互作用。 具体目标 1. 研究 DHPR oc1s 和 (5ia 亚基) 在体外与 wt-RyR1 和 RyR3 相互作用的能力。 RyR 将固定在能够支持脂质双层的 BIACORE 传感器芯片中，并通过在激活的表面上注射全长 a1s 或 p1a 亚基来监测 RyR1 与 DHPR 相互作用的特定结构域。我们将研究 (1s 和 (1a) 与包含多个 RyR1 区域的嵌合 RyR1/RyR3 受体库的结合特性。 RyR3 背景。具体目标 3. 定义参与与 RyR1 物理相互作用的 DHPR (1s 和 (1a) 的最小必要结构域。我们将测定嵌合 (1s/(1c 亚基) 与纯化的 RyR 体外相互作用的能力。具体目标 4. 研究已识别的 (1s/RyR1 和 (1a/RyR1 相互作用) 对 EC 偶联和逆行信号传导和静息 Ca2+ 的功能作用。一个广泛的库嵌合 RyR3/RyR1 和 DHPR（1s/(1c 构建体）将在单敲除和双敲除肌管（RyR、DHPR 和 RyR/DHPR 无效）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复杂信号传导过程对于开发旨在改善病理生理状态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.