MECHANISM OF INHIBITION OF CFTR BY AMP-ACTIVATED KINASE

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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.