NOVEL INTERACTION OF AMP-KINASE WITH CFTR C1 CHANNEL

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CFTR, the gene product which is defective in patients with cystic fibrosis (CF), functions not only as a C1- channel, but also as a coordinator and regulator of other membrane transporters in epithelia. Identifying and characterizing cellular proteins that interact with CFTR is necessary to understand the regulation of CFTR and its regulatory effects on other membrane transporters. This information could suggest new therapies for CF. This project will examine the consequences of a novel interaction we have discovered between CFTR and AMP-activated protein kinase (AMPK). AMPK is a kinase which is known to respond to the metabolic status of cells, phosphorylating and inhibiting several key biosynthetic enzymes under conditions of nutritional stress, thereby conserving cellular ATP. Its interaction with CFTR may be significant because CFTR is gated by ATP hydrolysis and may mediate ATP release from cells. The regions of both proteins that are important for the interaction will be determined using truncation mutants and the yeast two-hybrid system. AMPK-CFTR binding in vitro and in vivo will be explored through co-immunoprecipitation with epitope-tagged constructs, and conditions that modulate the strength of the interaction will be defined. The functional consequences of the AMPK-CFTR interaction will be studied by: examining the cellular expression and localization of AMPK in relation to CFTR, determining whether CFTR is a substrate for AMPK phosphorylation in vitro and in vivo, investigating the effects of this interaction on CFTR single channel properties and expression in cell membranes using patch-clamp techniques, and determining the effects of the AMPK-CFTR interaction on epithelial Na+ channel (ENaC) function.