Mechanisms of RyR1 Modulation by General Anesthetics

描述： 骨骼肌Ryanodine受体(RyR1)是细胞内钙离子的重要调节因子，促进 激励收缩耦合。这种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， 可能是致命的。麻醉剂调节该受体的分子内机制，或者即使 它们直接与RyR1相互作用，这是完全未知的。野生型和MH突变体(R615C) 猪RyR1，我们将使用光标记技术来确定是否和哪里有两种挥发性 麻醉剂，异氟醚和七氟醚，结合(目标1)；然后协调这些位置与结构和 冷冻电子显微镜下的结构变化(目标2)。我们将确定它们的单通道效应 利用平面脂质双层电生理学进行离子转运(目标3)。最后，我们将测量相对绑定亲和力 在预期的多个地点中，以及这些地点中哪些最有可能与功能效应有关，使用 复杂的计算方法(平衡分子动力学和炼金术自由能 摄动，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 单个站点，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，也在 异丙酚，一种注射用全身麻醉剂。RyR1由于其体积和 复杂性，但技术(光标、质谱学、低温电子显微镜、平面 双层电生理学、分子动力学)已经成熟到这种多学科 Campaign很有可能揭示麻醉剂调节的分子内机制。

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 .