Molecular Basis of Secretory Diarrhea

In secretory diarrhea, the protein mainly responsible for controlling electrolyte and water movement into the intestinal lumen is the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl- channel subject to regulation by protein kinase A (PKA) and protein kinase C (PKC). CFTR activation requires phosphorylation by PKA and ATP hydrolysis, events occurring in adjacent CFTR domains termed nucleotide binding domain 1 (nbd1) and the R domain. This project will use recombinant peptides to study how CFTR is regulated. Peptides containing CFTR(62O-830), termed RD's, will model the R domain, and peptides containing CFTR(417-830), termed NBD/RD's, will model the nbd1/R domain. Modified peptides will contain Ala or Asp substitutions at one or more Ser residues. The project has three aims: AIM #1 will use RD's to study how protein kinases act on the R domain. Phosphorylated RD's will be analyzed by tryptic mapping and HPLC/mass spectrometry to identify phosphorylation events accompanying maximal vs. partial stimulation of CFTR. Additional studies will examine the mechanism by which protein kinases act on RD's and will determine how synergism between protein kinases orchestrates the orderly phosphorylation of CFTR. AIM #2 will study how PKA controls the intrinsic function of the nbd1/R domain. Pilot studies indicate that NBD/RD's exhibit PKA-regulated ATPase activity. These peptides will be used to determine which RD phosphorylation events activate vs. inhibit the nbd1 ATPase, to test whether the regulation of nbd1 by PKA is affected either by PKC or by nbd2, and to test whether the nbd1 ATPase is affected by the deltaF5O8 mutation, the most common cause of cystic fibrosis (CF). Aim #3 will study how protein kinases control the ability of the R domain to activate the Cl- channel function of CFTR. RD's will be added to membrane patches containing a truncated CFTR lacking most of the R domain (deltaR/S66OA CFTR) to compare the ability of different modified RD's to regulate CF transport by CFTR. This project's long-term goal is to clarify how protein kinases act on the nbd1/R domain to regulate CFTR function. Detailed knowledge of CFTR regulation will be fundamental to developing pharmacological strategies to selectively augment or diminish CFTR function. Given the pivotal role of CFTR in the pathogenesis of diarrhea and CF this project has excellent prospects of leading to information of practical benefit in the treatment of these conditions.