Structure Function Studies of RyR1 in a Myogenic Knockout

RyR1 在肌源性敲除中的结构功能研究

• 批准号: 7100705
• 负责人: Paul D Allen
• 金额: $ 45.8万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-14 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7100705
• 关键词: binding sites; calcium channel; chimeric proteins; gene mutation; genetically modified animals; immunocytochemistry; laboratory mouse; oxidation reduction reaction; oxidative stress; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity; sarcoplasmic reticulum; site directed mutagenesis; thiols; voltage /patch clamp

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to identify the key regions within the primary sequence of theCa2+ release channel (ryanodine receptor; RyR1) of mammalian skeletal muscle sarcoplasmic reticulum (SR) which determine: 1. Sensitivity to physiologic activating and inhibiting cations, and 2. The ability of the channel to sense physiologic changes in local redox potential. Hypothesis I: Structural determinants of the cation activator and inhibitor sites are coordinated by spatially separated amino acids within the RyR1 linear sequence. Conserved negative charges within one or more of five cytoplasmic domains in the C-terminal -1000 amino acids of RyR1 coordinate cation binding and activation, whereas it appears that interactions between the C-terminal and central domains of RyR1, especially sequences encompassing difference regions D1 & D3, are needed to engage proper cation inhibition of RyRI. Specific Aim 1. Define the structural determinants in RyR1 that influence its binding and sensitivity to Ca2+ and Mg2+ using site-specific mutations of C-terminal truncated channels and full length RyR1 along with RyR1/RyR3 chimera. Specific Aim 2. Define the changes in cation binding constants and the activation energy (Ea) associated with channel activation for RyR1s with mutations within their cation binding motifs. Specific Aim 3. To define how altered cation regulation of RyR1 impacts excitation-contraction coupling and excitation-coupled Ca2+ entry. Hypothesis II: Hyper-reactive sulfhydryls in the primary structure of RyR1 constitute a trans- SR redox gradient sensor. Specific Aim 1. To mutate the six hyper-reactive thiols that we have discovered in RyR1 and define their contribution to trans SR redox sensing behavior. Specific Aim 2. To establish mechanistic links between redox state and the ability of cations to regulate RyR1 activity. The proposed studies will provide new information concerning the molecular mechanisms by which microsomal calcium channels residing within the SR/ER membrane are regulated by two important physiological parameters, cations (Ca2+and Mg2+) and cellular redox state. It is likely that these mechanisms are conserved among the multi-member family of Ca2+ release channels and may be significant in identifying new clinical targets for the prevention and treatment of injury resulting from oxidative stress.

描述（由申请人提供）：拟议的研究的长期目标是确定哺乳动物骨骼肌肌肉肌浆（SR）的theca2+释放通道（ryanodine受体； RyR1）的主要序列中的关键区域，这些区域确定了：1。对脉冲和2层次的脉冲和2层的敏感性敏感。假设I：阳离子活化剂和抑制剂位点的结构决定因素通过RYR1线性序列内的空间分离氨基酸协调。 RyR1坐标结合和激活的C端-1000氨基酸中五个细胞质结构域中的一个或多个中的保守负电荷，而似乎需要序列差异区域D1和D3之间的c-末端和中央域之间的相互作用，以使d1＆d3序列均适当地使Cation Cation Cation Cation Cation Cation Indiencation rundiention ribed ribed ryrii indribity ryyri indribity ryyri。具体目标1。定义RYR1中的结构决定因素，这些决定因素使用C末端截断通道的位点特异性突变以及RYR1/RYR3嵌合体以及全长RYR1的位点特异性突变影响其对Ca2+和Mg2+的敏感性。具体目标2。定义阳离子结合常数的变化以及与阳离子结合基序中突变的RyR1s通道激活相关的激活能量（EA）。具体目的3。定义RYR1的阳离子调节如何影响激发 - 收缩耦合和激发耦合的Ca2+进入。假设II：RYR1的主要结构中的高反应性硫ry烯构成了跨氧化还原梯度传感器。具体目标1。突变我们在RYR1中发现的六个高反应性硫醇，并定义它们对反式SR氧化还原传感行为的贡献。具体目的2。建立氧化还原状态与阳离子调节RYR1活性的能力之间的机械联系。拟议的研究将提供有关分子机制的新信息，该分子机制通过这些机制，通过这些机制，驻留在SR/ER膜内的微粒体钙通道受两个重要的生理参数，阳离子（Ca2+和MG2+）和细胞氧化还原状态调节。这些机制可能是在CA2+释放通道的多成员家族中保守的，并且可能在识别预防和治疗氧化应激引起的损伤的新临床目标方面很重要。