Regulation of KATP and Cl channel activity by AMPK, ATP and adenosine receptors: impact on membrane excitability during muscle activity and fatigue

值得注意的是，骨骼肌活动期间细胞膜离子通道活性的复杂变化。第一个变化发生在第1阶段，始于肌肉活动的开始，其特征是ClC-1 Cl-通道活性降低70%。第2阶段的特征是ClC-1和KATP通道活性大幅增加，达到刺激前水平的3倍。中度运动时，5 min内平均间质[K+]达到10 mM。我们对K+和Cl-对膜兴奋性的影响的研究提供了证据，表明第一阶段ClC-1通道活性的降低不仅对防止K+引起的力抑制很重要，而且实际上对K+在10 mM K+时增强力的能力也很重要。在第二阶段，ClC-1和KATP通道活性的增加被认为与疲劳有关，即当肌肉受到重复刺激时，力或功的减少。现在人们普遍认为肌肉疲劳的一个主要因素涉及肌浆网Ca2+释放的减少，这在一定程度上是由于膜兴奋性的降低。兴奋性的降低是由ClC-1活性的增加引起的，使K+效应从增强变为抑制，而通过KATP通道的K+外排的增加降低了动作电位的幅度，最终减少了节省力的ATP。***问题是，从肌肉活动开始（即第1阶段）到疲劳发展（即第2阶段），是什么控制了ClC-1和KATP通道活动的复杂变化。研究表明，运动开始时ClC-1通道活性的降低与PKC的磷酸化有关。我们也知道ATP受体的激活会降低ClC-1通道的活性，但这些ATP受体是否在运动开始时参与其中还有待确定。在膜片钳条件下，A1腺苷受体激活了KATP通道，但在疲劳状态下，A1受体是否参与了ClC-1和KATP通道的激活尚不清楚。在心肌中，重要的细胞能量传感器AMPK激活KATP通道；因此，它也可能激活骨骼肌疲劳期间的ClC-1和KATP通道。***长期目标是研究从肌肉活动开始到疲劳期间调节膜兴奋性的细胞内信号通路。本提案的短期目标是研究i)第一阶段ATP及其受体在优化肌肉性能中的作用；ii) AMPK和腺苷在触发疲劳中的作用（第二阶段）。提出了两种假说：1)“ATP受体通过降低ClC-1 Cl-通道活性、激活Na+ K+ ATP酶泵和促进K+诱导的力增强来优化肌肉性能”和2)“AMPK和腺苷通过降低泵活性和激活KATP和ClC-1通道来触发疲劳”。**

Noteworthy are complex changes in ion channel activity in the cell membrane during skeletal muscle activity. The first change, occurring during Phase 1, starts at the onset of muscle activity and is characterized by a 70% decreases in ClC-1 Cl- channel activity. Phase 2 occurs is characterized by large increases ClC-1 and KATP channel activity up to 3-times above the pre-stimulation level. During moderate exercise, mean interstitial [K+] reaches 10 mM within 5 min. Our studies on K+ and Cl- effects on membrane excitability are providing evidence that the reduction in ClC-1 channel activity during Phase 1 is important not only to prevent any K+-induced force depression, but in fact to augment the capacity of K+ to potentiate force at 10 mM K+. The increase in ClC-1 and KATP channel activity during Phase 2 is believed to be associated with fatigue, known as the decrease in force or work when muscles are repetitively stimulated. It is now well accepted that a major factor in muscle fatigue involves a decrease in Ca2+ release by the sarcoplasmic reticulum, which in turn is in part due to a decreased membrane excitability. The decreased excitability is triggered by increase in ClC-1 activity changing the K+ effect from a potentiation to a depression, while the increase in K+ efflux through KATP channels reduces action potential amplitude to eventually reduce force sparing ATP.***     The question is what controls the complex changes in the activity of ClC-1 and KATP channel activity from the onset of muscle activity (i.e., Phase 1) to the development of fatigue (i.e., Phase 2). It has been shown that the decrease in ClC-1 channel activity at the onset of exercise is related to a phosphorylation by PKC. It is also known that activation of ATP receptors reduce ClC-1 channel activity, but it remains to be determined whether these ATP receptors are involved at the onset of exercise. Under patch clamp conditions, the A1 adenosine receptors activate KATP channels, but it remains to be determined if the A1 receptors are involved in the activation of ClC-1 and KATP channels during fatigue. In cardiac muscle, AMPK, an important cell energy sensor, activate KATP channels; it is therefore possible that it also activates the ClC-1 and KATP channels during fatigue in skeletal muscle. ***     The long term objective is to study the intracellular signaling pathways that regulate membrane excitability from the onset of muscle activity to fatigue. The short term objectives of this proposal are to investigate i) the role of ATP and its receptors in optimizing muscle performance during Phase 1 and ii) the role of AMPK and adenosine in triggering fatigue (Phase 2). Two hypotheses are proposed: 1) "ATP receptors optimize muscle performance by lowering ClC-1 Cl- channel activity, activating Na+ K+ ATPase pump, and promoting K+-induced force potentiation" and 2) "AMPK and adenosine triggers fatigue by reducing the pump activity and activating KATP and ClC-1 channels". **