Activity of CFTR CL channels determines the composition of fluid that lines the airway surface. In cystic fibrosis (CF), the loss of this regulated channel disrupts normal electrolyte transport and may contribute directly to disease pathogenesis. Earlier work showed that the activity of CFTR CL channels in airway epithelia is regulated by changes in cellular levels of cAMP. An increase in cAMP leads to phosphorylation of CFTR's regulatory (R) domain which allows the channel to open and mediate transepithelial CL movement. This project focuses on the mechanisms by which the cytosolic R domain controls the CFTR channel. Preliminary data showed that addition of an R domain peptide (R1, containing 190 residues) to a CFTR variant in which much of the R domain had been deleted (CFTR-R/S660A) stimulated activity. However, un-phosphorylated R1 had not effect. These results show a novel mechanism of channel upregulation by a phosphorylated peptide. They also provide us with a unique system for studying the structure and function of the R domain and CFTR. The project will answer three question. 1) What parts of the R domain control the activity of CFTR? The investigators will test the hypothesis that smaller peptides from the R domain stimulate activity of CFTR-R/S660A. Conversely, they will delete smaller portions of the R domain from CFTR to learn how channel gating is altered. These two complimentary approaches will allow them to identify key functional and structural features of the domain to discover their effect on activity. 2) What is the structure of the R domain? We currently lack molecular level data about the three-dimensional structure of CFTR or any of its domains. Their preliminary data show that R domain peptide is soluble and functionally active. Thus, now is the time to solve the R domain structure. Guided by their functional studies, they will determine the high-resolution structure of functionally active R domain peptides using NMR spectroscopy. They will also test the exciting hypothesis that phosphorylation changes the structure. 3) How do alterations in R domain structure affect function? The information obtained in the first two specific aims will provide valuable framework of understanding and a model of the R domain. They will test that model using site-directed mutagenesis to address very specific questions about how structure determines function. The prospects for success in this project are enhanced by the combined electrophysiologic, biochemical, and structural skills of this unique team of investigators.
| Bartlett, Jennifer A; Meyerholz, David K; Wohlford-Lenane, Christine L et al. (2015) Increased susceptibility to otitis media in a Splunc1-deficient mouse model. Dis Model Mech 8:501-8 |
| Bartlett, Jennifer A; Albertolle, Matthew E; Wohlford-Lenane, Christine et al. (2013) Protein composition of bronchoalveolar lavage fluid and airway surface liquid from newborn pigs. Am J Physiol Lung Cell Mol Physiol 305:L256-66 |
| Reed, Matthew D; McCombie, Barbara E; Sivillo, Aimee E et al. (2012) Safety assessment of nebulized xylitol in beagle dogs. Inhal Toxicol 24:365-72 |
| Dong, Qian; Ostedgaard, Lynda S; Rogers, Christopher et al. (2012) Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-?F508 processing and alter its gating defect. Proc Natl Acad Sci U S A 109:917-22 |
| Pezzulo, Alejandro A; Starner, Timothy D; Scheetz, Todd E et al. (2011) The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol 300:L25-31 |
| Schmidt, Nathan W; Mishra, Abhijit; Lai, Ghee Hwee et al. (2011) Criterion for amino acid composition of defensins and antimicrobial peptides based on geometry of membrane destabilization. J Am Chem Soc 133:6720-7 |
| Bartlett, Jennifer A; Bartlett, Jennifer; Gakhar, Lokesh et al. (2011) PLUNC: a multifunctional surfactant of the airways. Biochem Soc Trans 39:1012-6 |
| Itani, Omar A; Chen, Jeng-Haur; Karp, Philip H et al. (2011) Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance. Proc Natl Acad Sci U S A 108:10260-5 |
| Fischer, Anthony J; Lennemann, Nicholas J; Krishnamurthy, Sateesh et al. (2011) Enhancement of respiratory mucosal antiviral defenses by the oxidation of iodide. Am J Respir Cell Mol Biol 45:874-81 |
| Gakhar, Lokesh; Bartlett, Jennifer A; Penterman, Jon et al. (2010) PLUNC is a novel airway surfactant protein with anti-biofilm activity. PLoS One 5:e9098 |
Showing the most recent 10 out of 77 publications
