Mechanisms of ENaC Regulation by AMP-activated Kinase

描述(由申请人提供)：母公司K08授予[DK59477]的目标一直是发现AMPK的潜在机制，AMPK是一种普遍存在的激酶，其活性与细胞代谢状态密切相关，它调节上皮细胞中CFTRCI通道的功能。这个项目已经产生了几个重要的发现。AMPK-CFTR的相互作用似乎在极化的肺和结肠上皮中具有生理学意义，并为离子转运与细胞代谢的耦合提供了一个新的范例。AMPK主要通过抑制CFTR的单通道开放概率而不是通过影响CFTR的质膜表达来调节CFTR的活性。AMPK依赖的CFTR抑制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，并且其他转运蛋白的活性与细胞代谢状态相耦合，AMPK可能通过调节cftr和/或独立于cftr来调节其他重要的转运蛋白。上皮性Na+通道(ENaC)通过调节肾脏远端肾单位对Na+的重吸收，在体内Na+和容量的动态平衡中发挥重要作用。在肺和其他组织中，ENAC功能也受到CFTR的调节，在囊性纤维化的发病机制中可能起着重要的作用。我们的初步数据表明，AMPK调节ENaC功能，潜在地将ENaC功能与代谢状态偶联。这项研究计划将扩大父母资助的重点，包括旨在阐明AMPK对非洲爪哇卵母细胞ENaC调节机制的研究。将仔细研究AMPK激活对ENaC活性影响的随时间变化的差异。短期和长期AMPK活性调节对ENaC质膜表达、转运和通道特性的影响也将被研究。最后，将寻求参与AMPK依赖的ENaC调控的潜在信号通路(S)。更好地理解AMPK对卵母细胞中ENaC的依赖调节机制将为未来极化上皮细胞和体内研究提供框架，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.