Mechanisms of RyR1 Modulation by General Anesthetics

全身麻醉药调节 RyR1 的机制

• 批准号: 10335174
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335174
• 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; Hot Spot; 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; Probability; Propofol; Protein Isoforms; Proteins; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcoplasmic Reticulum; Site; Site-Directed Mutagenesis; Skeletal Muscle; Specificity; Structure; Tacrolimus Binding Protein 1A; Technology; Testing; Validation; Work; adduct; antagonist; base; drug inhalation; 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: