Effects of MH mutations on function of dihydropyridine receptor

MH突变对二氢吡啶受体功能的影响

• 批准号: 8667317
• 负责人: KURT G BEAM
• 金额: $ 26.96万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-14 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8667317
• 关键词: Adult; Affect; Age; Anesthetics; Bioenergetics; Body Temperature; Calcium; Cell membrane; Cells; Charge; Collaborations; Coupling; Dantrolene; Dideoxy Chain Termination DNA Sequencing; Dihydropyridine Receptors; Elements; Energy Metabolism; Failure; Female; Fiber; Freeze Fracturing; Frequencies; Gender; General Anesthesia; Genetic; Heterogeneity; Human; Hypersensitivity; Hypersensitivity skin testing; Inborn Genetic Diseases; Inhalation Anesthetics; Inherited; Instruction; Intervention; Kinetics; Laboratories; Lead; Link; Lipid Peroxides; Malignant hyperpyrexia due to anesthesia; Measures; Membrane; Mitochondria; Modification; Molecular; Movement; Mus; Muscle; Muscle Cells; Muscle Fibers; Mutation; Nutrient; Pathogenesis; Pathology; Patients; Perioperative; Population; Post-Translational Protein Processing; Predisposition; Production; Proteins; Receptor Activation; Rest; RyR1; Ryanodine Receptor Calcium Release Channel; SERCA1; Sarcoplasmic Reticulum; Signal Transduction; Skeletal Muscle; System; Transducers; Transfection; Variant; aged; azumolene; base; exome; fluo-3-AM; insight; male; mutant; novel; patch clamp; prevent; rapid technique; research study; response; voltage

PROJECT SUMIV1ARY (See instructions): In skeletal muscle, the interaction between two proteins, the dihydropyridine receptor (DHPR) in the plasma membrane/transverse-tubules and the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR), is essential for linking electrical excitation to contraction (excitation-contraction coupling, EC coupling). In particular, it is thought that conformational changes of the DHPR (containing Cav1.1 as its principal subunit) in response to depolarization cause RyR1 to open and release calcium from the SR, and that this signaling depends on physical links between the two proteins. Significantly, mutations of the DHPR or RyR1 in humans can result in the inherited disorder of malignant hyperthermia susceptibility (MHS), whereby volatile anesthetics cause dysregulation of calcium release that can lead to a fatal rise in body temperature unless there is rapid intervention. With the long-term objective of understanding the interactions between the DHPR and RyR1, and the mechanisms of MH, the specific aims of Project 3 are: Aim 1.1: To use whole cell patch clamping to measure L-type Ca2+ current and charge movement (which arise directly from the DHPR), together with Ca release from the SR, to determine how MHS mutations (RyR1-R2435H and -T4826I; Cav1.1-R174W) in mouse and human myotubes affect bi-directional signaling. Aim 1.2: To determine the effect of dantrolene on bi-directional signaling in MHS myotubes. Aim 1.3: In collaboration with Core D, to determine if MHS mutations alter the frequency or disposition of DHPR tetrads in myotubes. Aim 2: To measure membrane currents and myoplasmic Ca2+ transients in dissociated FDB fibers from adult (3-6 mo) and aged (12-18 mo) male and female MHS mice (Cav1.1-R174W and RyR1-R163C,-R2435H, and -T4826I) to determine the heterogeneity that results from differences in gender, age and locus. Aim 3: To measure membrane currents and Ca2+ transients in FDB fibers from 3-6 month old male Het RyR1-T4826I MHS mice that have been crossed with mice over-expressing SERCA1 (enhanced SR Ca2+ filling) or dnTPRC6 (reduced SOCE), or which were administered 4-OH-BDE49 (reduced RyR1 leak) or salicylamine (γKA scavenger) to determine if modification of one of the 4 key elements associated with MHS can mitigate or abrogate alterations in RyR-DHPR bi-directional signaling. Aim 4.1: To determine whether Ca2+ currents, charge movements or voltage-gated Ca2+ transients are differentially affected by volatile anesthetics in WT or MHS mutant (RyR1-R1630, -R2435H, -T4826I; Cav1.1-R174W) FDBs. Aim 4.2: To determine whether effects of volatile anesthetics on Ca2+ channel function are prevented by treatment with dantrolene. Aim 5: To use expression in myotubes of proteins harboring MHS mutations newly discovered by Core C in order to determine their effects on bi-directional signaling and hypersensitivity to volatile anesthetics.

项目摘要（参见说明）： 在骨骼肌中，质膜/横管中的二氢吡啶受体 (DHPR) 和肌浆网 (SR) 中的 1 型兰尼碱受体 (RyR1) 两种蛋白质之间的相互作用对于连接电兴奋与收缩（兴奋-收缩耦合、EC 耦合）至关重要。特别是，人们认为 DHPR（包含 Cav1.1 作为其主要亚基）响应去极化而发生的构象变化导致 RyR1 打开并从 SR 中释放钙，并且这种信号传导取决于两种蛋白质之间的物理联系。值得注意的是，人类 DHPR 或 RyR1 的突变可导致恶性高热易感性 (MHS) 遗传性疾病，挥发性麻醉剂会导致钙释放失调，从而导致致命的体温升高，除非迅速干预。为了了解 DHPR 和 RyR1 之间的相互作用以及 MH 的机制，项目 3 的具体目标是： 目标 1.1：利用全细胞膜片钳测量 L 型 Ca2+ 电流和电荷运动（直接由 DHPR 产生），以及 SR 中的 Ca 释放，以确定 MHS 突变（RyR1-R2435H 和 -T4826I）如何发生； Cav1.1-R174W）在小鼠和人类肌管中影响双向信号传导。目标 1.2：确定丹曲林对 MHS 肌管双向信号传导的影响。目标 1.3：与 Core D 合作，确定 MHS 突变是否改变肌管中 DHPR 四分体的频率或分布。目标 2：测量成年（3-6 个月）和老年（12-18 个月）雄性和雌性 MHS 小鼠（Cav1.1-R174W 和 RyR1-R163C、-R2435H 和 -T4826I）分离的 FDB 纤维中的膜电流和肌质 Ca2+ 瞬变，以确定性别、年龄和位点差异导致的异质性。目标 3：测量 3-6 个月大的雄性 Het RyR1-T4826I MHS 小鼠 FDB 纤维中的膜电流和 Ca2+ 瞬变，这些小鼠已与过表达 SERCA1（增强 SR Ca2+ 填充）或 dnTPRC6（减少 SOCE）的小鼠杂交，或者施用 4-OH-BDE49（减少 RyR1 泄漏）或水杨胺（γKA） scavenger）以确定与 MHS 相关的 4 个关键元件之一的修饰是否可以减轻或消除 RyR-DHPR 双向信号传导的改变。目标 4.1：确定 WT 或 MHS 突变体（RyR1-R1630、-R2435H、-T4826I；Cav1.1-R174W）FDB 中的挥发性麻醉剂对 Ca2+ 电流、电荷运动或电压门控 Ca2+ 瞬变是否有不同影响。目标 4.2：确定丹曲林治疗是否可以防止挥发性麻醉药对 Ca2+ 通道功能的影响。目标 5：利用 Core C 新发现的含有 MHS 突变的蛋白质在肌管中的表达，以确定它们对双向信号传导和对挥发性麻醉剂过敏的影响。