Effects of MH mutations on function of dihydropyridine receptor

项目摘要（见说明）： 在骨骼肌中，质膜/横小管中的二氢吡啶受体（DHPR）和肌浆网（SR）中的1型兰尼碱受体（RyR 1）这两种蛋白质之间的相互作用对于将电兴奋与收缩联系起来（兴奋-收缩偶联，EC偶联）至关重要。特别是，据认为，DHPR（含有Cav1.1作为其主要亚基）的构象变化响应于去极化导致RyR 1打开并从SR释放钙，并且这种信号传导取决于两种蛋白质之间的物理连接。值得注意的是，人类DHPR或RyR 1的突变可能导致恶性高热易感性（MHS）的遗传性疾病，挥发性麻醉剂会导致钙释放失调，除非进行快速干预，否则可能导致体温致命性升高。本研究的长期目标是了解DHPR和RyR 1之间的相互作用以及MH的作用机制，具体目标为：目标1.1：利用全细胞膜片钳技术测量L型钙电流和电荷运动（直接由DHPR产生），以及SR中的Ca释放，以确定MHS突变如何影响细胞的生长。在小鼠和人肌管中的两种蛋白（RyR 1-R2435 H和-T4826 I; Cav1.1-R174 W）影响双向信号传导。目的1.2：确定丹曲林对MHS肌管中双向信号传导的影响。目的1.3：与核心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。目标二：测量成年（3-6月龄）和老年（12-18月龄）雄性和雌性MHS小鼠（Cav1.1-R174 W和RyR 1-R163 C、-R2435 H和-T4826 I）的游离FDB纤维中的膜电流和肌浆Ca 2+瞬变，以确定性别、年龄和基因座差异导致的异质性。目标3：为了测量来自3-6月龄雄性Het RyR 1-T4826 I MHS小鼠的FDB纤维中的膜电流和Ca 2+瞬变，所述小鼠已经与过表达SERCA 1的小鼠杂交（增强SR Ca 2+填充）或dnTPRC 6（减少SOCE），或被注射了4-OH-BDE 49（RyR 1泄漏减少）或水杨胺（γKA清除剂），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双定向信号目标4.1：确定WT或MHS突变体（RyR 1-R1630、-R2435 H、-T4826 I; Cav1.1-R174 W）FDB中挥发性麻醉剂对Ca 2+电流、电荷运动或电压门控Ca 2+瞬变的影响是否存在差异。目的4.2：确定丹曲林治疗是否可预防挥发性麻醉药对Ca 2+通道功能的影响。目标5：使用Core C新发现的携带MHS突变的蛋白质在肌管中的表达，以确定其对双向信号传导和对挥发性麻醉剂超敏反应的影响。

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.