The extracellular loops of ? and ? ENaC can be roteolytically cleaved at multiple sites by furin-type convertases, serine proteases and neutrophil elastase, leading to activation of the channel and increased Na+ absorption. In cystic fibrosis (CF) airways, ENaC is abnormally hyperactive due in part to excessive proteolytic cleavage, which contributes to a depletion of airway surface liquid (ASL) volume. This in turn may lead to mucus stasis and increased incidence of airway infections that frequently lead to the death of the patient. We have recently identified SPLUNC1 as a potent inhibitor of ENaC that binds to the extracellular side of ENaC and diminishes plasma membrane ENaC levels. We have also shown that when SPLUNC1 is knocked down by shRNA, normal human bronchial epithelial cultures can no longer regulate ENaC activity. We hypothesize that SPLUNC1 limits Na+ absorption by reducing ENaC surface densities, preventing exposure to extracellular proteases. We have identified the active site of SPLUNC1, and have synthesized an 18 amino acid peptide based on this site, called S18, which specifically binds to ?ENaC. Although S18 robustly inhibits ENaC in NL and CF airway epithelia, SPLUNC1 fails to regulate ENaC in CF airway epithelia. CF ASL is acidic due to the lack of CFTR-associated HCO3- secretion. Molecular modeling indicates that SPLUNC1 may undergo a pH- induced conformational change that """"""""buries"""""""" its active site, preventing binding to ENaC in the acidic CF ASL. Thus, we propose to (i) solve the crystal structure of SPLUNC1 to further refine this model, (ii) determine the mechanism whereby extracellular SPLUNC1 can reduce ENaC surface density and (iii) determine why SPLUNC1 fails to function in CF airways. S18-like peptides may be therapeutically beneficial in the treatment of CF lung disease. Furthermore, our data suggest that inhaled hypertonic HCO3- may offer significant advantages over hypertonic saline for restoring CF mucus clearance.

Public Health Relevance

CF mucus is dehydrated due to a genetically induced imbalance in salt and water transport in the lungs, leaving the airways prone to mucus plugging and recurrent infections. In this application, we propose to understand how CF airways can regulate salt and water transport to maintain mucus clearance. We also propose to develop novel peptides that can inhibit excessive salt absorption in CF airways to improve CF mucus hydration.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL108927-03
Application #
8645717
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Banks-Schlegel, Susan P
Project Start
2012-05-10
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
3
Fiscal Year
2014
Total Cost
$356,061
Indirect Cost
$111,061
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Garnett, James Peter; Kalsi, Kameljit K; Sobotta, Mirko et al. (2016) Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H(+) secretion. Sci Rep 6:37955
Walton, William G; Ahmad, Saira; Little, Michael S et al. (2016) Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1. Biochemistry 55:2979-91
Fellner, Robert C; Terryah, Shawn T; Tarran, Robert (2016) Inhaled protein/peptide-based therapies for respiratory disease. Mol Cell Pediatr 3:16
Ahmad, Saira; Tyrrell, Jean; Walton, William G et al. (2016) Short Palate, Lung, and Nasal Epithelial Clone 1 Has Antimicrobial and Antibiofilm Activities against the Burkholderia cepacia Complex. Antimicrob Agents Chemother 60:6003-12
Watson, Michael J; Lee, Shernita L; Marklew, Abigail J et al. (2016) The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor. Sci Rep 6:27390
Tyrrell, Jean; Qian, Xiaozhong; Freire, Jose et al. (2015) Roflumilast combined with adenosine increases mucosal hydration in human airway epithelial cultures after cigarette smoke exposure. Am J Physiol Lung Cell Mol Physiol 308:L1068-77
Ã…strand, Annika B M; Hemmerling, Martin; Root, James et al. (2015) Linking increased airway hydration, ciliary beating, and mucociliary clearance through ENaC inhibition. Am J Physiol Lung Cell Mol Physiol 308:L22-32
Xu, Xiaohua; Balsiger, Robert; Tyrrell, Jean et al. (2015) Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR. Biochim Biophys Acta 1850:1224-32
Choi, Hyun-Chul; Kim, Christine Seul Ki; Tarran, Robert (2015) Automated acquisition and analysis of airway surface liquid height by confocal microscopy. Am J Physiol Lung Cell Mol Physiol 309:L109-18
Ghosh, Arunava; Boucher, R C; Tarran, Robert (2015) Airway hydration and COPD. Cell Mol Life Sci 72:3637-52

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