Voltage sensor domain movements in skeletal muscle fiber activation

骨骼肌纤维的激活是所有身体运动的先决条件，是由肌肉启动的。 纤维动作电位(AP)。这种电去极化波沿着光纤传播，远离 神经肌肉连接和放射状进入横管(Tts)，导致膜带正电 Tt膜钙通道(Cav1.1)中的电压传感器结构域(VSD)通过 毗邻肌浆中骨骼肌兰尼定受体(RyR1)钙释放通道 网状膜。然而，TT VSD运动与SR RyR1释放的分子机制 人们对通道激活知之甚少，每个Cav1.1中的四个单独VSD的角色也不清楚 已经成立了。此外，在任何细胞中，还没有关于室间隔缺损运动对AP反应的研究 打字。在目标1中，我们首先确定AP期间VSD总电荷移动的时间进程(Q(t 波形，并将其与成人肌肉纤维中钙离子释放的时间进程(目标1)进行比较。在AIMS 2和3中 我们检查了在AP期间单个VSD运动的时间进程。我们比较了VSD时间 Q(T)的时间进程和通过RyR1激活SR钙释放的过程。VSD组件是 与测得的钙离子释放相比，明显缓慢或电压依赖性较低的钙离子释放将不能 激活RyR1钙释放通道。我们将描述之前发生的VSD组件的特征 并且与响应于AP的RyR1通道开放一致，因此是调节的候选 通道激活的效应器。在目标2中，我们使用Cys残基跟踪VSD的运动 Cav1.1靠近每个S4跨膜螺旋的胞外端，并呈荧光反应。在AIM 3我们使用在细胞质末端附近或跨膜内引入的人工荧光氨基酸。 S4片段本身或在Cav1.1αI-II和II-III细胞质环中被认为对Cav1.1-RyR1至关重要 耦合。在目标4中，我们通过实验确定了VSD电荷消除突变的效果 导致人类疾病(低血钾性周期性瘫痪或恶性高热)。我们用的是高速 (&lt；50微米S/行)含荧光染色或荧光残留物的纤维的行扫描共聚焦成像 或在每个VSD中。我们还将使用钙离子指示器来监测钙离子信号并计算潜在的钙离子 在单个接入点期间，在完整的电压钳制光纤中从SR释放通量。我们的研究将阐明基本的 骨骼肌钙释放调控的分子机制及Cav1.1电压传感器的作用 在低钾周期性麻痹和恶性高热中发生突变的电荷。这个项目有 对肌肉的基础膜生物物理和通道激活的即时高影响，对多个 学科和长期的潜力，以进一步了解问题的病理生理学问题 运动和呼吸都是各种晚期疾病状态和衰老的共同特征。

Activation of skeletal muscle fibers, which is a prerequisite for all bodily movements, is initiated by the muscle fiber action potential (AP). This wave of electrical depolarization spreads along the fiber away from the neuromuscular junction and radially into the transverse tubules (TTs), causing positively charged membrane voltage sensor domains (VSDs) in the TT membrane Ca2+ channel (Cav1.1) to trigger Ca2+ release via the abutting skeletal muscle ryanodine receptor (RyR1) Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. However, the molecular mechanisms coupling TT VSD movements to SR RyR1 release channel activation are poorly understood, and the roles of the four individual VSDs within each Cav1.1 are not established. Furthermore, there are no previous studies of VSD movement in response to an AP in any cell type. Here in Aim 1 we first determine the time course (Q(t)) of total VSD charge movement during the AP waveform, and compare it to the time course of Ca2+ release (Aim 1) in adult muscle fibers. In Aims 2 and 3 we examine the time course of the individual VSD movements during an AP. We compare the VSD time courses to the time course of Q(t) and of activation of SR Ca2+ release via RyR1. VSD components that are obviously slow or less voltage dependent compared to the measured Ca2+ release would not be capable of activating the RyR1 Ca2+ release channel. We will characterize the VSD components that do occur prior to and coincident with RyR1 channel opening in response to an AP, and are thus candidates for regulatory effectors of channel activation. In Aim 2 we track VSD movements using cys residues introduced individually in Cav1.1 near the extracellular end of each of the S4 transmembrane helices and fluorescently reacted. In Aim 3 we use artificial fluorescent amino acids introduced near the cytoplasmic end or within the transmembrane S4 segment itself or in the Cav1.1 alpha I-II and II-III cytoplasmic loops considered critical for Cav1.1-RyR1 coupling. In Aim 4 we experimentally determine the effects of charge-eliminating mutations of the VSDs which cause human diseases (either hypokalemic periodic paralysis or malignant hyperthermia). We use high speed (<50 µs/line) line-scan confocal imaging of fibers containing fluorescently stained or fluorescent residues near or in each VSD. We will also use Ca2+ indicators to monitor Ca2+ signals and calculate the underlying Ca2+ release flux from the SR during a single AP in intact voltage clamped fibers. Our studies will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of Cav1.1 voltage sensor charges that are mutated in hypokalemic periodic paralysis and malignant hyperthermia. This project has immediate high impact for basic membrane biophysics of muscle and channel activation, for multiple disciplines and in the long-term the potential to further our understanding of the pathophysiology of problems of both locomotion and breathing common to a variety of advanced diseased states and aging.