Cystic fibrosis (CF) is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. In humans and pigs, the loss of CFTR impairs respiratory host defenses, causing airway infection. But CF mice are spared. We found that in all three species, CFTR secreted bicarbonate into airway surface liquid. In humans and pigs lacking CFTR, unchecked H+ secretion by the nongastric H+/K+ adenosine triphosphatase (ATP12A) acidified airway surface liquid, which impaired airway host defenses. In contrast, mouse airways expressed little ATP12A and secreted minimal H+; consequently, airway surface liquid in CF and non-CF mice had similar pH. Inhibiting ATP12A reversed host defense abnormalities in human and pig airways. Conversely, expressing ATP12A in CF mouse airways acidified airway surface liquid, impaired defenses, and increased airway bacteria. These findings help explain why CF mice are protected from infection and nominate ATP12A as a potential therapeutic target for CF.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The most common mutation, a deletion of the phenylalanine at position 508 (⌬F508), disrupts processing of the protein. Nearly all human CFTR-⌬F508 is retained in the endoplasmic reticulum and degraded, preventing maturation to the plasma membrane. In addition, the F508 deletion reduces the activity of single CFTR channels. Human CFTR-⌬F508 has been extensively studied to better understand its defects. Here, we adopted a cross-species comparative approach, examining human, pig, and mouse CFTR-⌬F508. As with human CFTR-⌬F508, the ⌬F508 mutation reduced the single-channel activity of the pig and mouse channels. However, the mutant pig and mouse proteins were at least partially processed like their wild-type counterparts. Moreover, pig and mouse CFTR-⌬F508 partially restored transepithelial Cl ؊ transport to CF airway epithelia. Our data, combined with earlier work, suggest that there is a gradient in the severity of the CFTR-⌬F508 processing defect, with human more severe than pig or mouse. These findings may explain some previously puzzling observations in CF mice, they have important implications for evaluation of potential therapeutics, and they suggest new strategies for discovering the mechanisms that disrupt processing of human CFTR-⌬F508.airway epithelia ͉ chloride transport ͉ cystic fibrosis ͉ mouse models

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption.

Cyclosporin A (CsA), a powerful immunosuppressive drug, inhibits the synthesis of lymphokines in T lymphocytes at the level of gene transcription. Using protein extracts from El4 lymphoma cells we show that the binding of lymphocyte‐specific factors interacting with the two so‐called purine boxes (Pu‐boxes) of the interleukin 2 (IL‐2) enhancer are missing in CsA‐treated cells. The CsA‐sensitive factors are newly synthesized upon induction. The most prominent factor consists of 45 kd polypeptides and contacts both Pu‐boxes at the two central G residues within the identical core sequence AAGAGGAAAA. The CsA‐mediated suppression of factor binding to the Pu‐boxes correlates well with functional studies in which the inducible, T cell‐restricted proto‐enhancer activity of Pu‐boxes was selectively repressed by CsA. These observations support the conclusion that the suppression of factor binding to the Pu‐boxes by CsA impairs the activity of IL‐2 and of further lymphokine genes, thereby inhibiting the synthesis of lymphokines in T lymphocytes.

Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-⌬F508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and ⌬F508 channels. Adding curcumin also increased Cl ؊ transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl ؊ channels.

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In developing gene therapy for cystic fibrosis (CF) airways disease, a transgene encoding a partially deleted CF transmembrane conductance regulator (CFTR) Cl ؊ channel could be of value for vectors such as adeno-associated virus that have a limited packaging capacity. Earlier studies in heterologous cells indicated that the CFTR R (regulatory) domain is predominantly random coil and that parts of the R domain can be deleted without abolishing channel function. Therefore, we designed a series of CFTR variants with shortened R domains (between residues 708 and 835) and expressed them in well-differentiated cultures of CF airway epithelia. All of the variants showed normal targeting to the apical membrane, and for the constructs we tested, biosynthesis was like wild type. Moreover, all constructs generated transepithelial Cl ؊ current in CF epithelia. Comparison of the Cl ؊ transport suggested that the length of the R domain, the presence of phosphorylation sites, and other factors contribute to channel activity. A variant deleting residues 708 -759 complemented CF airway epithelia to the same extent as wild-type CFTR and showed no current in the absence of cAMP stimulation. In addition, expression in nasal mucosa of CF mice corrected the Cl ؊ transport defect. These data provide insight into the structure and function of the R domain and identify regions that can be deleted with retention of function. Thus they suggest a strategy for shortening the transgene used in CF gene therapy.

To better understand the function of the conserved C terminus of the cystic fibrosis (CF) transmembrane conductance regulator, we studied constructs containing deletions in the C-terminal tail. When expressed in well differentiated CF airway epithelia, each construct localized predominantly to the apical membrane and generated transepithelial Cl ؊ current. The results suggested that neither the C-terminal PSD-95͞Discs-large͞ZO-1 (PDZ)-interacting motif nor other C-terminal sequences were absolutely required for apical expression in airway epithelia. Surprisingly, deleting an acidic cluster near the C terminus reduced both channel opening rate and transepithelial Cl ؊ transport, indicating that it influences channel gating. These results may help explain the relative paucity of CF-associated mutations in the C terminus.