MECHANISM OF INHIBITION OF CFTR BY AMP-ACTIVATED KINASE

描述（改编自应用程序） 哈洛斯博士目前正在实验室进行研究 宾夕法尼亚大学的福斯基特博士他的职业目标是 该赠款的期限是进一步发展技术专长（例如， 在分子、生物化学和电生理学方法学中）， 成功的独立调查职业所必需的创造性技能。 经过1-2年的指导研究奖学金培训，他计划 在肾脏科的终身教职职位上过渡。 长期职业目标是成为一名完全独立的学术研究者 在一个实验室结果可以被 应用于肾脏学广泛领域内的重要临床问题 和细胞运输生理学;特别是，调节和协调 上皮运输系统的一部分。这个研究项目的基础是一本小说 在我们的实验室中发现的CFTR， ATP调节的Cl-通道在囊性纤维化中有缺陷， 代谢传感器AMP活化蛋白激酶（AMPK）。使用酵母 双杂交和生物化学测定，C-末端调控结构域的 AMPK的α（催化）亚基被证明与CFTR的C-末端结合 尾巴AMPK与CFTR共定位于大鼠上皮细胞的顶端分布 组织和体外磷酸化CFTR。此外，AMPK与 非洲爪蟾卵母细胞CFTR抑制cAMP激活的CFTR全细胞电导， 高达50%。由于AMPK对细胞ATP的变化有反应， AMPK抑制CFTR可能是抑制CFTR的机制 在代谢应激的条件下，这表明了一种新的范式， 离子转运与细胞代谢的耦合。这项研究的目的是 项目的目的是确定AMPK依赖性CFTR抑制的机制。的 AMPK激活对单通道特性（Po）和表面的影响 将在表达CFTR的上皮细胞中测定CFTR的表达（N）。 因为α-AMPK与CFTR的结合及其激酶活性似乎都是 需要赋予这种抑制，进一步的目标是调查 这些现象如何转化为CFTR抑制的分子细节， 在CFTR C-末端的大分子复合物的背景。最终目标 将AMPK功能效应的研究扩展到运输系统 与CFTR密切相关并受CFTR调节。

DESCRIPTION (adapted from the application) Dr. Hallows is currently conducting his research fellowship in the laboratory of Dr. Foskett at the University of Pennsylvania. His career goals for the period of this grant are to further develop both the technical expertise (e.g., in molecular, biochemical and electrophysiological methodologies) and the creative skills necessary for a successful independent investigative career. After another 1-2 years of mentored research fellowship training, he plans to make the transition to a tenure-track faculty position within a renal division. Long-term career goals are to become a fully independent academic investigator performing basic research in an environment where bench results could be applied to important clinical problems within the broad fields of nephrology and cellular transport physiology; especially, the regulation and coordination of epithelial transport systems. The basis for this research project is a novel interaction that was discovered in our laboratory between CFTR, the ATP-regulated Cl-channel which is defective in cystic fibrosis, and the metabolic sensor AMP-activated protein kinase (AMPK). Using both yeast two-hybrid and biochemical assays, the C-terminal regulatory domain of the alpha (catalytic) subunit of AMPK was shown to bind to the CFTR C-terminal tail. AMPK co-localized in an apical distribution with CFTR in rat epithelial tissues and phosphorylated CFTR in vitro. Moreover, AMPK co-expression with CFTR in Xenopus oocytes inhibited cAMP-activated CFTR whole-cell conductance by up to 50 percent. Because AMPK responds to changes in cellular ATP, this inhibition of CFTR by AMPK may serve as a mechanism for inhibition of CFTR under conditions of metabolic stress, which suggests a novel paradigm for the coupling of ion transport to cellular metabolism. The goals of this research project are to determine the mechanisms for AMPK-dependent CFTR inhibition. The effects of AMPK activation on single-channel properties (Po) and surface expression (N) of CFTR will be determined in CFTR-expressing epithelial cells. Because both binding of alpha-AMPK to CFTR and its kinase activity appear to be required to confer this inhibition, further goals are to investigate the molecular details of how these phenomena translate into CFTR inhibition within the context of the macromolecular complex at the CFTR C-terminus. A final goal is to extend the search for functional effects of AMPK to transport systems that are closely associated with and modulated by CFTR.