Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)2− anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 μm Au(CN)2−, a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Relative Au(CN)2− permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337‐S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent.

Transepithelial anion secretion in many tissues depends upon the activity of basolateral channels. Using monolayers of the Calu‐3 cell line, a human submucosal serous cell model mounted in an Ussing chamber apparatus, we investigated the nature of the K+ channels involved in basal, cAMP‐ and Ca2+‐stimulated anion secretion, as reflected by the transepithelial short circuit current (Isc). The non‐specific K+ channel inhibitor Ba2+ inhibited the basal Isc by either 77 or 16 % when applied directly to the basolateral or apical membranes, respectively, indicating that a basolateral K+ conductance is required for maintenance of basal anion secretion. Using the K+ channel blockers clofilium and clotrimazole, we found basal Isc to be sensitive to clofilium, with a small clotrimazole‐sensitive component. By stimulating the cAMP and Ca2+ pathways, we determined that cAMP‐stimulated anion secretion was almost entirely abolished by clofilium, but insensitive to clotrimazole. In contrast, the Ca2+‐stimulated response was sensitive to both clofilium and clotrimazole. Thus, pharmacologically distinct basolateral K+ channels are differentially involved in the control of anion secretion under different conditions. Isolation of the basolateral K+ conductance in permeabilized monolayers revealed a small basal and forskolin‐stimulated Isc. Finally, using the reverse transcriptase‐polymerase chain reaction, we found that Calu‐3 cells express the K+ channel genes KCNN4 and KCNQ1 and the subunits KCNE2 and KCNE3. We conclude that while KCNN4 contributes to Ca2+‐activated anion secretion by Calu‐3 cells, basal and cAMP‐activated secretion are more critically dependent on other K+ channel types, possibly involving one or more class of KCNQ1‐containing channel complexes.

Exposure to reactive oxygen species (ROS) is associated with tissue damage in the lung and may be a common element in the pathogenesis of all inflammatory lung diseases. Exposure to the ROS hydrogen peroxide (H2O2) evoked a rapid increase in transepithelial anion secretion across monolayers of the human submucosal gland serous cell line Calu‐3. This increase was almost entirely abolished by the addition of diphenylamine‐2‐carboxylate (DPC), implicating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel in the response. The response was also reduced by inhibitors of basolateral K+ channels. Studies of electrically isolated apical and basolateral membranes revealed that H2O2 stimulated both apical Cl− and basolateral K+ conductances (GCl and GK). Apical GCl was sensitive to DPC, but unaffected by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), suggesting that CFTR is the major anion conduction pathway mediating the response to H2O2. Additionally, H2O2 had no effect on GCl in the presence of the adenylate cyclase inhibitor SQ22536 or following maximal stimulation of GCl with forskolin, implicating the cAMP‐dependent protein kinase pathway in the apical response to H2O2. Basolateral GK was reduced by the K+ channel inhibitors clotrimazole and clofilium, indicating roles for KCNN4 and KCNQ1 in the H2O2‐stimulated response. We propose that ROS‐stimulated anion secretion from serous cells plays an important role in keeping the airways clear from damaging radicals that could potentially initiate tissue destruction. Our finding that this response is CFTR dependent suggests that an important host defence mechanism would be dysfunctional in the cystic fibrosis (CF) lung. Loss of this compensatory protective mechanism could expose the CF lung to ROS for extended periods, which could be important in the pathogenesis of CF lung disease.

The present study was aimed at investigating the innate susceptibility of C57BL/6-Cftrunc/Cftrunc knockout [B6-Cftr (-/-)] mice to pulmonary infection with Pseudomonas aeruginosa. Our results indicate that 58.4% of B6-Cftr (-/-) mice died within 6 d following lung infection with 10(5) P. aeruginosa entrapped in agar beads, whereas only 12.1% of B6-Cftr (+/+) mice died over the same period of time. Moreover, the number of bacteria recovered from the lungs of B6-Cftr (-/-) mice 3 and 6 d after infection was significantly higher than that observed in their littermate controls. No correlation was found between the weight or age of the animals and the number of viable bacteria recovered from the lungs of mice. Histopathological examination of lung sections from P. aeruginosa-infected mice revealed that the infection results in a severe bronchopneumonia. Both B6-Cftr (-/-) knockout mice and their littermate controls developed similar lung pathology during the course of infection. Overall, results reported in the present study suggest that a defect at the Cftr locus leads to an exacerbation of P. aeruginosa lung infection resulting in a dramatically increased mortality rate and higher bacterial load.