Mechanisms of ENaC Regulation by AMP-activated Kinase

描述（由申请人提供）：亲本K08基金[DK59477]的目标是发现AMPK的潜在机制，AMPK是一种无处不在的激酶，其活性与细胞代谢状态密切相关，可调节上皮细胞中CFTR CI-通道的功能。这个项目已经产生了几个重要的发现。AMPK-CFTR相互作用似乎在极化肺和结肠上皮中具有生理相关性，并为离子转运与细胞代谢的耦合提供了新的范例。AMPK主要通过抑制CFTR的单通道打开概率来调节CFTR活性，而不是通过影响CFTR的质膜表达。ampk依赖性抑制CFTR的分子细节现在也变得更加清晰。由于CFTR调节其他上皮转运蛋白，并且由于其他转运蛋白的活性与细胞代谢状态耦合，AMPK可能通过其对CFTR和/或独立于CFTR的调节，潜在地调节其他重要的转运蛋白。上皮Na+通道（ENaC）通过调节远端肾单位的肾Na+重吸收，在全身Na+和体积稳态中起关键作用。肺和其他组织中的EnaC功能也受到CFTR的调节，并可能在囊性纤维化的发病机制中发挥重要作用。我们的初步数据表明AMPK调节ENaC功能，可能将ENaC功能与代谢状态耦合起来。该研究项目将扩大父母资助的重点，包括旨在阐明非洲爪蟾卵母细胞中依赖ampk的ENaC调节机制的研究。我们将仔细研究AMPK活化对ENaC活性影响的时间依赖性差异。短期和长期AMPK活性调节对ENaC质膜表达、运输和通道特性的影响也将被研究。最后，将寻求涉及ENaC的ampk依赖性调节的潜在信号通路。更好地了解卵母细胞中依赖ampk的ENaC调控机制，将为未来极化上皮细胞和体内的研究提供框架，并为肾脏的盐和水运输如何与代谢状态耦合以及缺血性和缺氧性肾损伤的发病机制提供新的见解。

DESCRIPTION (provided by applicant): The goals of the parent K08 grant [DK59477] have been to discover the underlying mechanisms by which AMPK, a ubiquitous kinase whose activity is finely tuned to cellular metabolic status, modulates the function of the CFTR CI- channel in epithelial cells. This project has already yielded several important findings. The AMPK-CFTR interaction appears to be physiologically relevant in polarized lung and colonic epithelia and provides a new paradigm for the coupling of ion transport to cellular metabolism. AMPK regulates CFTR activity predominantly through an inhibition in the single-channel open probability of CFTR rather than through effects on CFTR plasma membrane expression. The molecular details underlying the AMPK-dependent inhibition of CFTR are also now becoming clearer. Because CFTR regulates other epithelial transport proteins and because the activity of other transport proteins are coupled to cellular metabolic status, AMPK may potentially regulate other important transport proteins, both through its regulation of CFTR and/or independently of CFTR. The epithelial Na+ channel (ENaC) plays a critical role in total-body Na+ and volume homeostasis by regulating renal Na+ reabsorption in the distal nephron. EnaC function is also modulated by CFTR in the lung and other tissues and may play an important rote in the pathogenesis of cystic fibrosis. Our preliminary data suggest that AMPK regulates ENaC function, potentially coupling ENaC function to metabolic status. This research program will expand the focus of the parent grant to include studies designed to elucidate the mechanisms for AMPK-dependent regulation of ENaC in Xenopus oocytes. Time-dependent differences in the effects of AMPK activation on ENaC activity will be closely examined. The effects of short- and long-term AMPK activity modulation on ENaC plasma membrane expression, trafficking, and channel properties will also be studied. Finally, the underlying signaling pathway(s) involved in AMPK-dependent regulation of ENaC will be sought. A better understanding of the mechanisms for AMPK-dependent regulation of ENaC in oocytes should provide the framework for future studies in polarized epithelial cells and in vivo and yield new insights into how salt and water transport by the kidney is coupled to metabolic state and into the pathogenesis of ischemic and hypoxic renal injury.