Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating

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DESCRIPTION (provided by applicant): This application proposes a career development program for Dr. Christoph O. Randak to develop into an independent physician-scientist and academic teacher and mentor. The heart of the proposal is an intensive laboratory training experience to develop scientific skills and understanding in protein chemistry and biochemistry of membrane proteins. The cystic fibrosis transmembrane conductance regulator (CFTR), a regulated chloride channel, is a representative of one of the largest families of membrane proteins, the adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Mutations in the gene encoding CFTR cause cystic fibrosis (CF). Understanding the molecular mechanisms of CFTR channel gating will facilitate the development of means to cure and control CF. Many previous studies have focused on CFTR's ATPase activity and how it governs CFTR gating. Recent work has shown that CFTR also interacts with adenosine 5'-monophosphate (AMP) and that adenylate kinase activity (ATP:AMP phosphotransfer) can gate CFTR. The objective of this research proposal is to identify key mechanisms and key amino acid residues involved in AMP-binding and adenylate kinase activity of CFTR. Using labeling techniques with azido-nucleotides and CFTR patch-clamping, the hypothesis will be tested that AMP and ATP interact cooperatively with CFTR, and that AMP prevents ATP hydrolysis and induces ATP:AMP phosphotr-ansfer. The results are expected to elucidate how ATP and AMP mutually influence their interaction with CFTR, and how AMP influences CFTR's enzymatic activity; it is also expected to identify key residues involved in AMP-binding to CFTR. The program will provide Dr. Randak with formal career mentoring and guidance. Dr. Michael J. Welsh will assume responsibility as mentor and Drs. Jeffrey C. Murray, Paul B. McCray Jr., and Kevin P. Campbell will be advisors. Additional objectives are to increase Dr. Randak's teaching and mentoring experience, to guide him in establishing a research laboratory, to increase his skills in scientific speaking and writing, to help him expand his contacts and collaborations with other scientists, and to prepare him to apply for independent research funding. The University of Iowa will provide a state-of-art research environment dedicated to Dr. Randak's scientific development and academic success. RELEVANCE (See instructions): Knowledge from these studies will provide a framework to develop activators and inhibitors of CFTR channel function. Such compounds may be useful for the treatment of diseases associated with too little (cystic fibrosis) or too much (secretory diarrhea) CFTR chloride currents. Because CFTR is an ABC transporter, this knowledge may also impact treatments of other ABC transporter-related diseases. (End of Abstract)