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
基本信息
- 批准号:10586027
- 负责人:
- 金额:$ 33.99万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:Action PotentialsAdultAgingAmino AcidsBiophysicsBreathingCalciumCalcium ionChargeClosure by clampCouplingCytoplasmDisciplineDiseaseEnvironmentFiberFluorometryFunctional disorderHousingHypokalemic periodic paralysisIndividualIon ChannelLabelLinkLocationLocomotionMalignant hyperpyrexia due to anesthesiaMammalian CellMeasurementMeasuresMediatingMembraneMolecularMonitorMovementMuscleMuscle FibersMutateMutationNeuromuscular JunctionOocytesPhysiologicalPropertyPublicationsRadialReactionReagentRoleRyanodine Receptor Calcium Release ChannelSarcoplasmic ReticulumScanningSignal TransductionSkeletal MuscleSpeedStainsStimulusSystemTimeVariantadvanced diseasecell typeconfocal imagingdisease-causing mutationelectrical measurementextracellularhuman diseaseinterestmolecular domainreceptor couplingresponsesensorvoltagevoltage clampvoltage gated channel
项目摘要
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沿着纤维传播远离
神经肌肉接头和径向进入横小管(TT),导致带正电荷的膜
TT膜Ca 2+通道(Cav1.1)中的电压传感器域(VSD),以触发Ca 2+通过
邻近骨骼肌肌浆中的RyR 1钙释放通道
网状膜。然而,将TT VSD运动与SR RyR 1释放耦合的分子机制
通道激活知之甚少,每个Cav1.1中四个单独VSD的作用也不清楚。
确立了习此外,以前没有研究室间隔缺损运动对任何细胞中AP的反应
类型.在这里,在目标1中,我们首先确定AP期间总VSD电荷移动的时间过程(Q(t)),
波形,并将其与成人肌纤维中Ca 2+释放的时间过程(Aim 1)进行比较。目标2和3
我们检查了在AP期间各个VSD运动的时间过程。我们比较室间隔缺损时间
Q(t)和通过RyR 1激活SR Ca 2+释放的时间过程。VSD组件,
与测量的Ca 2+释放相比,明显缓慢或较少的电压依赖性将不能
激活RyR 1钙释放通道。我们将描述在手术前确实发生的室间隔缺损的特征。
并且与响应于AP的RyR 1通道开放一致,因此是调控的候选者。
通道激活的效应器。在目标2中,我们使用在细胞中单独引入的cys残基追踪VSD运动。
Cav1.1在每个S4跨膜螺旋的细胞外末端附近,并发生荧光反应。在Aim中
3我们使用人工荧光氨基酸引入细胞质末端附近或跨膜内
S4片段本身或Cav1.1 α I-II和II-III胞质环中被认为对Cav1.1-RyR 1至关重要
偶合器.在目标4中,我们通过实验确定VSD的电荷消除突变的影响,
导致人类疾病(低钾性周期性麻痹或恶性高热)。我们用高速
(<50 µs/line)线扫描共聚焦成像,用于包含荧光染色或荧光残留物的纤维,
或在每个VSD中。我们还将使用Ca 2+指标来监测Ca 2+信号并计算潜在的Ca 2 +
在完整电压箝位光纤中的单个AP期间从SR释放通量。我们的研究将阐明
骨骼肌钙释放调控的分子机制及Cav1.1电压传感器的作用
在低钾性周期性麻痹和恶性高热中发生突变的电荷。这个项目
立即对肌肉和通道激活的基本膜生物物理学产生高度影响,
学科和长期的潜力,以进一步了解我们的问题的病理生理学,
运动和呼吸都是各种晚期疾病和衰老的共同特征。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Erick Omar Hernandez-Ochoa其他文献
Erick Omar Hernandez-Ochoa的其他文献
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{{ truncateString('Erick Omar Hernandez-Ochoa', 18)}}的其他基金
Rad and amyotrophic lateral sclerosis (ALS)
放射线和肌萎缩侧索硬化症 (ALS)
- 批准号:
10400852 - 财政年份:2018
- 资助金额:
$ 33.99万 - 项目类别:
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