Transport mechanisms and airway surface layer function in upper respiratory epithelium, and the effect of flexible loop mutations on CFTR function

Abstract

Cystic fibrosis (CF) is caused by mutations on the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), which results in defective protein processing and channel gating. Almost 90% of CF patients carry the ΔF508 mutation, but it is uncertain why this causes defects in CFTR protein processing and channel gating.

I investigated the role of the H3-H4 loop in the dysfunction of ΔF508-CFTR using CFTR single-residue-deletion mutants (residues 503-513). Cells transfected with wild-type (WT), ΔV510- or ΔS511-CFTR expressed mature glycosylated CFTR, whereas other deletion mutants displayed the processing defect producing immature less glycosylated protein. Low-temperature culture enhanced protein maturation only in ΔF508- and ΔY512-CFTR, which also exhibited low open probability and prolonged interburst interval compared to WT-CFTR. These data suggest that the ΔF508 and ΔY512 mutations may impair CFTR protein processing and channel activity by similar mechanisms.

To test whether the CFTR processing defects of ΔF508 or ΔY512 mutations resulted from altered residual function of the H3-H4 loop structure, I constructed glycine- and alanine-substituted mutations in the loop structure of ΔF508- and ΔY512-CFTR which would increase loop flexibility. The ratio of mature:total protein expression in ΔF508-CFTR was enhanced by V510G and the double mutation G509A/V510G. Conversely, mutations V510A/S511A did not promote protein expression of ΔY512-CFTR. Single-channel activities of V510G/G509A/ΔF508-CFTR and V510A/S511A/ΔY512-CFTR were increased compared with those of ΔF508- and ΔY512-CFTR, respectively. As the loop mutations largely rescued the gating and processing defects of ΔF508-CFTR, the data suggest that loop dysfunction may be an important defect that underlies the functional abnormalities of ΔF508- CFTR.

The surface of airway epithelia is covered with airway surface liquid (ASL), containing antimicrobial substances for killing invaded bacteria. In CF patients, mucociliary clearance and bacterial killing in the airway is impaired. However, the role of CFTR in regulating antimicrobial substances remains uncertain.

Both the apical surface of cultured pig tracheal epithelia (A-EPI) and collected ASL exhibited bactericidal activity against Pseudomonas aeruginosa. ASL contained several antimicrobial substances, including lactoferrin, LPLUNC1 and lysozyme. The bacterial killing rate was positively correlated with the amount of <10 kDa protein, suggesting that antimicrobial peptides might be important in airway epithelial bacterial killing.

ASL bacterial killing was maximal at pH 7.4, and diminished at higher or lower pH; HCO3− was not essential for bacterial killing on the A-EPI. Inhibition of CFTR or ENaC/Na+/H+-exchanger decreased bacterial killing in the ASL, but not the concentration of antimicrobial substances, suggesting that decreased antimicrobial activity due to the lowered pH may underlie impaired bacterial killing in CF patients. ASL bacterial killing was not directly affected by Na+ concentration or ionic strength.

Basolateral Na+/K+-ATPase (NKA) is suggested to act as the Na+ sensor in respiratory epithelial cells, but the mechanism by which NKA regulates Na+ transport is uncertain. Inhibition of NKA activity by 60 nM ouabain or zero K+ medium for 7 hours decreased transepithelial Na+ and Cl– transport. Ouabain increased NKA and ENaC-mRNA. The PI-3K, PKC and ERK1/2 (but not Src or AMPK) signaling pathways may be involved in the ouabain-induced reduction in transepithelial Na+ transport.