Mechanism and function of a novel purinergic signaling cascade in skeletal muscle

骨骼肌中新型嘌呤能信号级联的机制和功能

• 批准号: 8267585
• 负责人: Andrew Alvin Voss
• 金额: $ 10.88万
• 依托单位: CALIFORNIA STATE POLY U POMONA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8267585
• 关键词: 9-anthroic acid; Acetates; Action Potentials; Affect; Amphibia; Antibodies; Binding; Biochemical; Carboxylic Acids; Cell Culture Techniques; Chloride Channels; Chloride Ion; Chlorides; Data; Development; Disease; Dyes; Electrophysiology (science); Enzyme-Linked Immunosorbent Assay; Event; Exercise; Fatigue; Fiber; Frequencies; Generations; Goals; Human; Individual; Isoenzymes; Knock-out; Length; Measurement; Measures; Membrane; Methods; Microelectrodes; Modeling; Molecular; Mus; Muscle; Muscle Fatigue; Muscle Fibers; Muscle function; Mutation; Myotonia Congenita; Optics; Pathway interactions; Patients; Permeability; Phospho-Specific Antibodies; Physiological; Physiology; Preparation; Protein Isoforms; Protein Kinase C; Protein Kinase C Inhibitor; Rattus; Receptor Activation; Regulation; Relative (related person); Reporting; Research; Resistance; Rest; Role; Sarcolemma; Signal Pathway; Signal Transduction; Signaling Molecule; Skeletal Muscle; Staining method; Stains; Staurosporine; Surface; Symptoms; System; Techniques; Testing; Transgenic Organisms; Tubular formation; Western Blotting; Wild Type Mouse; electrical measurement; electrical property; extracellular; flexor digitorum brevis; inhibitor/antagonist; insight; novel; phorbol-12-myristate; purinoceptor P2Y1; research study; response; voltage clamp

DESCRIPTION (provided by applicant): Currently, the molecular events in skeletal muscle that underlie exercise-induced fatigue, changes during disuse, and the symptoms of diseases such as myotonia congenita are not fully understood. For example, in disuse experiments, the chloride permeability of muscle has been shown to increase as a result of reduced protein kinase C activity, but the signaling events that induce those changes are unknown. The long- term goal of the research in this proposal is to elucidate physiological and pathophysiological mechanisms of muscle adaptation. This will be achieved by examining a novel purinergic signaling cascade discovered by the PI. The discovery of this signaling cascade was surprising, as it had been known since 1969 that human skeletal muscle releases ATP during exercise. The mechanism likely went undetected because it is unique to mammalian muscle relative to amphibian and most of the previous examinations used amphibian muscle or cell culture preparations. The recent study by the PI revealed that physiologically relevant levels of extracellular ATP act on P2Y1 receptors to rapidly (seconds to minutes) inhibit chloride channels in mammalian skeletal muscle. Because chloride channels are responsible for most of the resting conductance in skeletal muscle, this discovery has significant implications for the physiology of muscle excitability and fatigue. Pathologically, mutations in ClC-1, by far the predominant muscle chloride channel and the likely target of P2Y1 receptors, underlie the hyperexcitability seen in patients with Thomsen and Becker myotonias. Moreover, recent reports suggest that muscle chloride channels regulate the onset of exercise-related fatigue. The aims of the research in this proposal are to characterize further the P2Y1/chloride channel signaling mechanism and to determine the effects of this cascade on active electrical properties in muscle. Electrophysiological, biochemical and pharmacological techniques will be used in Specific Aims 1 & 2 to determine whether ClC-1 and protein kinase C, a known regulator of ClC-1, function in the P2Y1/chloride channel pathway. Optical and electrophysiological methods will be used in Specific Aim 3 to measure the effects of chloride channel inhibition by P2Y1 receptors on the propagation of action potentials in the sarcolemma and transverse tubular system. By providing mechanistic insights and examining the physiological role of a novel purinergic signaling cascade, the results from the proposed studies will have implications for exercise-related muscle fatigue, muscle disuse, and disorders such as myotonia congenita. PUBLIC HEALTH RELEVANCE: The focus of this proposal is to examine a novel cell signaling cascade in mammalian skeletal muscle that is activated by extracellular ATP and results in the inhibition of chloride channels. The proposed research aims to identify key signaling molecules that participate in the mechanism and to determine the role of this signaling cascade in active muscle. This research will provide insights into the physiological role of ATP release during muscle activity and it has implications for exercise-induced fatigue, muscle disuse, and treating disorders such as myotonia congenita.

描述（由申请人提供）：目前，骨骼肌中导致运动性疲劳的分子事件、废用期间的变化以及先天性肌强直等疾病的症状尚未完全了解。例如，在废用实验中，由于蛋白激酶C活性降低，肌肉的氯渗透性增加，但诱导这些变化的信号事件尚不清楚。本研究的长期目标是阐明肌肉适应的生理和病理生理机制。这将通过检查PI发现的一种新的嘌呤能信号级联来实现。这种信号级联的发现是令人惊讶的，因为自1969年以来，人类骨骼肌在运动期间释放ATP。该机制可能未被发现，因为它是哺乳动物肌肉相对于两栖动物所特有的，并且大多数以前的检查使用两栖动物肌肉或细胞培养制剂。PI最近的研究表明，生理相关水平的细胞外ATP作用于P2 Y1受体，以快速（秒至分钟）抑制哺乳动物骨骼肌中的氯离子通道。由于氯离子通道负责骨骼肌中的大多数静息电导，因此这一发现对肌肉兴奋性和疲劳的生理学具有重要意义。在病理学上，ClC-1突变是迄今为止主要的肌肉氯离子通道和P2 Y1受体的可能靶点，它是在Alcohsen和Becker肌强直患者中观察到的过度兴奋的基础。此外，最近的报告表明，肌肉氯离子通道调节运动相关疲劳的发生。本研究的目的是进一步表征P2 Y1/氯离子通道信号机制，并确定该级联对肌肉主动电特性的影响。电生理学、生物化学和药理学技术将用于特定目标1和2，以确定ClC-1和蛋白激酶C（一种已知的ClC-1调节剂）是否在P2 Y1/氯离子通道途径中起作用。在具体目标3中将使用光学和电生理学方法来测量P2 Y1受体对氯离子通道的抑制对肌膜和横管系统中动作电位传播的影响。通过提供机制的见解和检查一种新的嘌呤能信号级联的生理作用，从拟议的研究结果将有运动相关的肌肉疲劳，肌肉废用，和疾病，如先天性肌强直的影响。 公共卫生相关性：该建议的重点是研究哺乳动物骨骼肌中由细胞外ATP激活并导致氯离子通道抑制的新型细胞信号级联。拟议的研究旨在确定参与该机制的关键信号分子，并确定这种信号级联在活动肌肉中的作用。这项研究将为肌肉活动期间ATP释放的生理作用提供见解，并对运动引起的疲劳，肌肉废用和治疗先天性肌强直等疾病具有影响。