In cystic fibrosis (CF), loss of the CFTR CI- channel disrupts airway epithelial electrolyte transport, thereby contributing directly to the disease pathogenesis. Two processes control the activity of the CFTR CI-channel: phosphorylation of its R domain and ATP interactions with its nucleotide-binding domains (NBDs). This project focuses on these two regulatory mechanisms. 1. Regulation of CFTR CI- channel activity by phosphorylation motifs in the R domain. Our recent structural and functional studies suggest that R domain phosphorylation motifs and their flanking residues are central to regulation. We will probe CFTR function with phosphorylated and unphosphorylated R domain peptides comprising these motifs. This novel strategy will reveal mechanisms by which the R domain regulates CFTR. 2. Control of CFTR CI- channels by adenylate kinase activity. For a long time we have known that the CFTR can hydrolyze ATP (ATP++ADP+Pi). Our preliminary data suggest that in addition to its ATPase activity, CFTR has adenylate kinase activity (ATP+AMP'2ADP). We will test how this novel enzymatic activity controls channel activity and identify responsible mechanisms. By using powerful new approaches and by testing novel hypotheses, the results of this work will give us an unprecedented look at how CFTR works. As we focus on very specific structural elements and enzymatic mechanisms, our data will help explain earlier results and will yield exciting new insight into the relationship between CFTR structure and function. The results will have substantial implications for the function of other medically important ABC transporters and for the development of new treatments for CF.