Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Relative to wild type CFTR, the most common mutation deltaF508-CFTR, present in >85% of CF patients is expressed at low levels in the plasma membrane and has reduced in vivo activity. Thus to correct the deltaF508-CFTR mutation both the number of plasma membrane channels and the activity of individual channels must be increased. Our data suggest that the reduced in vivo activity of deltaF508 - CFTR is due to a reduced level of phosphorylation. In this study we will test two hypotheses, 1) that differences in CFTR activity can be caused by differences in channel phosphorylation, and 2) that differences in the activity of wild type and deltaF508-CFTR are due to differences in phosphorylation. By activating CFTR under various experimental conditions and by examining CFTR with serine to alanine mutations at phosphorylation sites, we will generate CFTR with different phosphorylation states. Then by comparing the channel kinetics of different phosphorylation states, we will generate structure function relationships. Our studies will provide. biochemical evidence for the hypotheses that PKC-dependent phosphorylation is required for PKA-dependent activation and that protein phosphatases show selectivity with respect sites of dephosphorylation. These studies will assist in the development of drugs to treat CF patients with deltaF508-CFTR.
In specific aim I, two-dimensional peptide mapping, site-directed mutagenesis and mass spectroscopy will be used to identify sites of PKC- and PKA-dependent CFTR phosphorylation and establish experimental conditions where CFTR phosphorylation differs.
In specific aim II, channel kinetic parameters will be determined for CFTR that has been phosphorylated on different sites. Alterations in mean open and closed times, Po and the number of active channels will be correlated with phosphorylation at specific sites.
In specific aim III, we will compare phosphorylation and channel kinetics of wild type and deltaF508-CFTR to determine if differences in channel activity are correlated with differences in phosphorylation.
| Vais, Horia; Gao, Guang-Ping; Yang, Michael et al. (2004) Novel adenoviral vectors coding for GFP-tagged wtCFTR and deltaF508-CFTR: characterization of expression and electrophysiological properties in A549 cells. Pflugers Arch 449:278-87 |
| Vais, Horia; Zhang, Rugang; Reenstra, William W (2004) Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation. Am J Physiol Cell Physiol 287:C737-45 |
