MECHANISM OF INHIBITION OF CFTR BY AMP-ACTIVATED KINASE

说明(改编自应用程序) 哈洛斯博士目前正在实验室进行他的研究 宾夕法尼亚大学的福斯基特博士。他的职业生涯目标是 这笔赠款的期限是为了进一步发展技术专长(例如， 分子、生物化学和电生理学方法)和 成功的独立调查事业所必需的创造性技能。 在再接受一到两年的有指导的研究奖学金培训后，他计划 过渡到肾脏部门的终身教职。 长期的职业目标是成为一名完全独立的学术调查员 在一个环境中进行基础研究，在这种环境中，可能会有 在广泛的肾脏病领域中应用于重要的临床问题 和细胞运输生理学；特别是调节和协调 上皮性运输系统。这项研究项目的基础是一项新的 在我们的实验室中发现的CFTR、 ATP调节的氯离子通道在囊性纤维化中的缺陷，以及 代谢感受器AMP激活的蛋白激酶(AMPK)。同时使用两种酵母 双杂交和生化分析，C端的调节域 AMPK的α(催化)亚单位与CFTRC-末端结合 尾巴。AMPK与CFTR共定位于大鼠上皮细胞的顶端分布 组织和体外磷酸化的CFTR。此外，与AMPK共表达 非洲爪哇卵母细胞CFTR抑制cAMP激活的CFTR全细胞电导 高达50%。由于AMPK对细胞内ATP的变化做出反应，因此 AMPK对CFTR的抑制作用可能是其作用机制之一 在代谢应激条件下，这提出了一种新的范式 离子转运与细胞新陈代谢的耦合。这项研究的目标是 项目的目的是确定AMPK依赖的CFTR抑制的机制。这个 AMPK激活对单通道特性(Po)和表面的影响 CFTR的表达(N)将在表达CFTR的上皮细胞中确定。 因为α-AMPK与CFTR的结合和它的激酶活性似乎都是 需要授予这种抑制，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 CFTRC-末端的大分子复合体的上下文。最终目标 是将AMPK的功能效应研究扩展到交通系统 与cftr密切相关并受cftr调控。

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.