Effects of MH mutations on function of dihydropyridine receptor

项目SUMIV1ARY(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型兰诺定受体(RyR1)之间的相互作用是连接电子兴奋与收缩(兴奋-收缩偶联，EC偶联)的关键。特别是，DHPR(以Cav1.1为主要亚基)在去极化反应中的构象变化被认为导致RyR1开放并从SR释放钙，并且这一信号依赖于两个蛋白质之间的物理联系。值得注意的是，人类DHPR或RyR1的突变可能导致遗传性恶性高热易感性(MHS)疾病，即挥发性麻醉剂导致钙释放失调，除非迅速干预，否则可能导致致命的体温上升。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：使用全细胞膜片钳技术测量直接来自DHPR的R1Se的L型钙电流和电荷移动，以及SR的钙释放，以确定MHS突变(RyR1R2435H和-T4826I；Cav1.1-R174W)如何影响双向信号转导。目的：研究丹曲林对MHS肌管双向信号转导的影响。目标1.3：与核心D合作，确定MHS突变是否改变了DHPR四分体在肌管中的频率或分布。目的：测定成年(3-6mo)和老年(12-18mo)雄性和雌性MHS小鼠(Cav1.1-R174W和RyR1-R163C、-R2435H和-T4826I)游离FDB纤维的膜电流和肌浆钙瞬变，以确定性别、年龄和基因座差异所导致的异质性。目的：测定3-6月龄雄性Het RyR1T4826I MHS小鼠FDB纤维的膜电流和钙瞬变，并与高表达SERCA1(增强型SR钙充盈)或dnTPRC6(降低SOCE)的小鼠杂交，或给予4-OH-BDE49(减少RyR1泄漏)或水杨胺(YKA清除剂)，以确定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双向信号的改变。目的：研究WT和MHS突变体(RyR1-R1630，-R2435H，-T4826I；Cav1.1-R174W)的钙电流、电荷运动或电压门控性钙瞬变是否受到挥发性麻醉药的不同影响。目的4.2：探讨丹曲林能否阻止挥发性麻醉药对钙通道功能的影响。目的：利用Core C新发现的含有MHS突变的蛋白在肌管中的表达，以确定其对双向信号传导和对挥发性麻醉药的超敏反应的影响。

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 elect R1cal 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 a R1se directly from the DHPR), together with Ca release from the SR, to determine how MHS mutations (RyR1R2435H 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 Ca^* transients in FDB fibers from 3-6 month old male Het RyR1T4826I 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 (YKA 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 Ca^* currents, charge movements or voltage-gated Ca^* 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.