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，尚未完全了解骨骼肌的分子事件，这些分子事件是运动引起的疲劳，废弃期间的变化以及诸如Myotonia congenatia之类的疾病症状的基础。例如，在废弃实验中，由于蛋白激酶C活性的降低，肌肉的氯化物通透性已显示出增加，但是诱导这些变化的信号传导事件尚不清楚。该提案中该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发现的新型嘌呤能信号传导级联反应来实现。这种信号传导级联的发现令人惊讶，因为自1969年以来就知道人类骨骼肌在运动过程中释放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而独特的，并且以前的大多数检查都使用了两栖动物肌肉或细胞培养的制剂。 PI的最新研究表明，生理上相关的细胞外ATP水平对P2Y1受体作用，可快速（秒至分钟）抑制哺乳动物骨骼肌中的氯化物通道。由于氯化物通道负责骨骼肌中的大多数静息电导，因此该发现对肌肉兴奋性和疲劳的生理具有重要意义。从病理上讲，CLC-1中的突变是迄今为止主要的肌肉氯化物通道和P2Y1受体的可能靶标，这是Thomsen和Becker Myotonias患者的过度兴奋性。此外，最近的报道表明，肌肉氯化物通道调节与运动相关的疲劳的发作。该提案中该研究的目的是进一步表征P2Y1/氯化物通道信号传导机制，并确定该级联反应对肌肉中主动电性能的影响。电生理，生化和药理学技术将用于特定的目标1和2，以确定CLC-1和蛋白激酶C（CLC-1的已知调节剂）在P2Y1/氯化物通道途径中的功能是否功能。光学和电生理方法将用于特定目标3中，以测量P2Y1受体抑制氯化物通道对肌膜和横向管状系统中作用电位传播的影响。通过提供机械洞察力并检查新型嘌呤能信号级联的生理作用，拟议研究的结果将对与运动相关的肌肉疲劳，肌肉消失以及诸如Myotonia congenita等疾病产生影响。 公共卫生相关性：该提案的重点是检查哺乳动物骨骼肌肉中新型的细胞信号传导级联反应，该骨骼肌肉被细胞外ATP激活，并导致抑制氯化物通道。拟议的研究旨在确定参与机制的关键信号分子，并确定该信号级联在活性肌肉中的作用。这项研究将提供对ATP释放在肌肉活动期间的生理作用的见解，并且对运动引起的疲劳，肌肉消失和治疗疾病（如Myotonia congenita）具有影响。