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

描述(申请人提供)：目前，骨骼肌中导致运动性疲劳的分子事件、停用过程中的变化以及先天性肌强直等疾病的症状还不完全清楚。例如，在废用实验中，由于蛋白激酶C活性的降低，肌肉的氯离子渗透性已经被证明是增加的，但引起这些变化的信号事件尚不清楚。这项研究的长期目标是阐明肌肉适应的生理和病理生理学机制。这将通过检查PI发现的一种新的嘌呤能信号级联来实现。这一信号级联的发现令人惊讶，因为自1969年以来，人们就知道人类骨骼肌在运动时会释放三磷酸腺苷。这种机制可能没有被发现，因为它是哺乳动物肌肉相对于两栖动物特有的，而且以前的大多数检查使用的是两栖动物肌肉或细胞培养制剂。PI最近的研究表明，生理上相关水平的细胞外ATP作用于P2Y1受体，迅速(几秒到几分钟)抑制哺乳动物骨骼肌的氯通道。由于氯离子通道负责骨骼肌的大部分静息电导，这一发现对肌肉兴奋性和疲劳的生理学具有重要意义。病理上，ClC-1的突变是Thomsen和Becker肌强直患者过度兴奋的基础，ClC-1是到目前为止主要的肌肉氯通道和P2Y1受体的可能靶标。此外，最近的报道表明，肌肉氯通道调节运动性疲劳的发生。这项研究的目的是进一步研究P2Y1/氯通道的信号机制，并确定这一级联反应对肌肉活动电特性的影响。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和已知的ClC-1调节因子蛋白激酶C是否在P2Y1/氯通道通路中发挥作用。在具体目标3中，将使用光学和电生理方法来测量P2Y1受体抑制氯通道对肌膜和横管系统动作电位传播的影响。通过提供机制洞察和研究一种新的嘌呤能信号级联的生理作用，拟议的研究结果将对运动相关的肌肉疲劳、肌肉停用和先天性肌强直等疾病产生影响。

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.