Voltage sensor domain movements in skeletal muscle fiber activation

骨骼肌纤维激活中的电压传感器域运动

• 批准号: 10116082
• 负责人: MARTIN F SCHNEIDER
• 金额: $ 33.99万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-10 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10116082
• 关键词: Action Potentials; Adult; Aging; Amino Acids; Biophysics; Breathing; Calcium; Calcium ion; Charge; Closure by clamp; Coupling; Discipline; Disease; Environment; Fiber; Fluorometry; Functional disorder; Housing; Hypokalemic periodic paralysis; Individual; Ion Channel; Label; Link; Location; Locomotion; Malignant hyperpyrexia due to anesthesia; Mammalian Cell; Measurement; Measures; Mediating; Membrane; Molecular; Monitor; Movement; Muscle; Muscle Fibers; Mutate; Mutation; Neuromuscular Junction; Oocytes; Physiological; Property; Publications; Radial; Reagent; Role; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Scanning; Signal Transduction; Skeletal Muscle; Speed; Stains; Stimulus; System; Time; Variant; advanced disease; cell type; confocal imaging; disease-causing mutation; electrical measurement; extracellular; human disease; interest; molecular domain; receptor coupling; response; sensor; voltage; voltage clamp; voltage gated channel

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.

骨骼肌肉纤维的激活是所有身体运动的先决条件，是由肌肉引发的 纤维动作电位（AP）。这种电去极化的波沿着纤维散布 神经肌肉连接并从根本上进入横向管（TTS），导致带正电的膜 TT膜Ca2+通道（CAV1.1）中的电压传感器域（VSD）触发Ca2+​​通过 固定骨骼肌ryanodine受体（RYR1）Ca2+释放通道 网状膜。但是，将TT VSD运动耦合到SR RYR1释放的分子机制 通道激活的理解很少，并且每个CAV1.1中四个单独的VSD的作用不是 已确立的。此外，以前没有对任何细胞中AP的VSD运动的研究 类型。在AIM 1中，我们首先确定AP期间总VSD电荷运动的时间过程（Q（t）） 波形，并将其与成年肌肉纤维中Ca2+释放（目标1）的时间过程进行比较。在目标2和3中 我们检查了AP期间单个VSD运动的时间过程。我们比较VSD时间 Q（t）的时间过程和通过RYR1释放SR Ca2+激活的课程。 VSD组件 显然，与测量的Ca2+释放相比，缓慢或更少的电压无法 激活RYR1 Ca2+释放通道。我们将表征在此之前确实发生的VSD组件 与RYR1通道响应于AP的开放，因此是调节性的候选者 通道激活的影响。在AIM 2中，我们使用单独引入的CYS残差跟踪VSD运动 Cav1.1附近S4跨膜螺旋的细胞外端，并荧光反应。目标 3我们使用胞质端附近或跨膜内引入的人造荧光氨基酸 S4段本身或CAV1.1 alpha I-II和II-III II胞质环被认为对CAV1.1-RYR1至关重要 耦合。在AIM 4中，我们通过实验确定VSD的电荷突变的影响 引起人类疾病（降低性周期性瘫痪或恶性高温）。我们使用高速 （<50 µs/line）线扫描的纤维胶合成像，这些纤维包含荧光染色或荧光残差附近 或在每个VSD中。我们还将使用CA2+指示器监视CA2+信号并计算基础CA2+ 在完整的电压夹紧纤维中，在单个AP期间从SR释放通量。我们的研究将阐明基本 调查骨骼肌中Ca2+释放的分子机制和CAV1.1电压传感器的作用 在降低性周期性瘫痪和恶性高温中突变的电荷。这个项目有 对肌肉和通道激活的基本膜生物物理学的直接影响 学科以及长期的潜力，使我们进一步理解了问题的病理生理学 各种晚期患病状态和衰老常见的运动和呼吸。