Mechanisms of RyR1 Modulation by General Anesthetics

全身麻醉药调节 RyR1 的机制

• 批准号: 10544782
• 负责人: Roderic G Eckenhoff
• 金额: $ 43.78万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-10 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544782
• 关键词: Affect; Affinity; Agonist; Alzheimer' s Disease; Anesthetics; Binding; Binding Sites; Biological Assay; Caffeine; Calcium; Calmodulin; Cell physiology; Clinical; Coupling; Cryoelectron Microscopy; Data; Development; Disease; Electrophysiology (science); Equilibrium; Exposure to; Family suidae; Floods; Free Energy; General anesthetic drugs; Genes; Goals; Human; Image; Individual; Injectable; Ions; Isoflurane; Length; Life; Ligand Binding; Ligands; Link; Lipid Bilayers; Location; Malignant hyperpyrexia due to anesthesia; Mass Spectrum Analysis; Molecular Conformation; Muscle Cells; Mutagenesis; Mutate; Mutation; Neurodegenerative Disorders; Operative Surgical Procedures; Outcome; Parents; Pathologic; Pathology; Patients; Persons; Pharmaceutical Preparations; Photoaffinity Labels; Predisposition; Probability; Propofol; Protein Isoforms; Proteins; Reticulum; Ryanodine Receptor Calcium Release Channel; Sarcoplasm; Site; Site-Directed Mutagenesis; Skeletal Muscle; Specificity; Structure; Tacrolimus Binding Protein 1A; Technology; Testing; Validation; Volatilization; Work; adduct; antagonist; drug inhalation; endoplasm; experimental study; in silico; molecular dynamics; multidisciplinary; mutant; novel; particle; porcine stress syndrome; receptor; sevoflurane; simulation; three dimensional structure

Description: The skeletal muscle ryanodine receptor (RyR1) is a critical regulator of cellular calcium to facilitate excitation contraction coupling. Mutations in this receptor underlie a number of serious disorders, one of which is called malignant hyperthermia (MH). MH typically occurs unexpectedly during a general anesthetic, and can be fatal. The intramolecular mechanism by which anesthetics modulate this receptor, or even if they interact directly with RyR1, is completely unknown. For both wild type and MH mutant (R615C) porcine RyR1, we will use photolabeling technology to determine whether and where two volatile anesthetics, isoflurane and sevoflurane, bind (Aim 1); then reconcile these sites with the structure and structural changes using cryo-electron microscopy (Aim 2). We will determine their single-channel effects on ion transit using planar lipid bilayer electrophysiology (Aim 3). Finally, we will gauge relative binding affinity among the expected multiple sites and which of these are most likely to be linked to functional effects, using sophisticated computational approaches (equilibrium molecular dynamics and alchemical free energy perturbation, Aim 4). We will test the effect of site-directed and natural mutations on binding affinity in individual sites, which will then be iteratively tested by experiment in Aims 1 and 3. The included preliminary data lends confidence for a successful outcome in each aim, and also has yielded surprising results in the case of propofol, an injectable general anesthetic. The RyR1 is a challenging target due to its size and complexity, but the technologies (photolabeling, mass spectrometry, cryo-electron microscopy, planar bilayer electrophysiology, molecular dynamics) have matured to a point where this multidisciplinary campaign has a very high probability of revealing the intramolecular mechanism of anesthetic modulation .

描述： 骨骼肌兰尼碱受体 (RyR1) 是细胞钙的关键调节因子，可促进 激励收缩耦合。该受体的突变是许多严重疾病的基础，其中之一 这被称为恶性高热（MH）。 MH 通常在全身麻醉期间意外发生， 并且可能是致命的。麻醉剂调节该受体的分子内机制，或者即使 它们与 RyR1 直接相互作用，是完全未知的。对于野生型和 MH 突变体 (R615C) 猪RyR1，我们将使用光标记技术来确定两个是否以及在何处挥发 麻醉剂、异氟烷和七氟烷，结合（目标 1）；然后将这些站点与结构相协调 使用冷冻电子显微镜观察结构变化（目标 2）。我们将确定它们对单通道的影响 使用平面脂质双层电生理学进行离子传输（目标 3）。最后，我们将测量相对结合亲和力 在预期的多个站点中，以及其中哪些最有可能与功能效果相关，使用 复杂的计算方法（平衡分子动力学和炼金术自由能 扰动，目标 4)。我们将测试定点突变和自然突变对结合亲和力的影响 各个站点，然后将通过目标 1 和 3 中的实验进行迭代测试。 数据为每个目标的成功结果提供了信心，并且在以下方面也取得了令人惊讶的结果 丙泊酚是一种注射全身麻醉剂。 RyR1 是一个具有挑战性的目标，因为它的尺寸和 复杂性，但技术（光标记、质谱、冷冻电子显微镜、平面 双层电生理学、分子动力学）已经成熟到了这种多学科的程度 该运动很有可能揭示麻醉调节的分子内机制。