The cystic fibrosis transmembrane conductance regulator. Effects of the most common cystic fibrosis-causing mutation on the secondary structure and stability of a synthetic peptide.

Deletion of phenylalanine 508 (delta Phe-508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein causes approximately 70% of all cases of cystic fibrosis. This residue lies in a region of the protein that we have synthesized chemically and shown to bind adenine nucleotides (Thomas, P. J., Shenbagamurthi, P., Ysern, X., and Pedersen, P. L. (1991) Science 251, 555-557). A peptide lacking this critical residue, but otherwise corresponding to this crucial part of the protein, now also has been chemically synthesized and purified. This mutant peptide (P-66) exhibits a significant loss of beta-sheet structure as compared with the wild type peptide (P-67). Furthermore, urea denaturation of peptide structure reveals that P-66 is less stable than P-67. Although under non-denaturing conditions both peptides bind adenine nucleotides with high affinity, the loss of structural stability is reflected in the binding function of the peptides. Thus, P-67, in contrast to P-66, retains a significant capacity for nucleotide binding in 4 M urea. These results suggest a model for impaired delta Phe-508 CFTR function.     

Domain-domain associations in cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is caused by mutations inthe cystic fibrosis transmembrane conductance regulator (CFTR) gene.CFTR is a chloride channel whose activity requires protein kinaseA-dependent phosphorylation of an intracellular regulatory domain(R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs).To identify potential sites of domain-domain interaction within CFTR,we expressed, purified, and refolded histidine (His)- andglutathione-S-transferase (GST)-tagged cytoplasmic domainsof CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated bymeasuring tryptophan fluorescence quenching. Trypticdigestion of in vitro phosphorylated his-NBD1-R and in situphosphorylated CFTR generated the same phosphopeptides. An interactionbetween NBD1-R and NBD2 was assayed by tryptophan fluorescencequenching. Binding among all pairwise combinations of R-domain, NBD1,and NBD2 was demonstrated with an overlay assay. To identifyspecific sites of interaction between domains of CFTR, an overlay assaywas used to probe an overlapping peptide library spanning allintracellular regions of CFTR with his-NBD1, his-NBD2, andGST-R-domain. By mapping peptides from NBD1 and NBD2 that bound toother intracellular domains onto crystal structures for HisP, MalK, andRad50, probable sites of interaction between NBD1 and NBD2 wereidentified. Our data support a model where NBDs form dimers with theATP-binding sites at the domain-domain interface.

     

Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.     

Annexin V is directly involved in cystic fibrosis transmembrane conductance regulator's chloride channel function

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel, which is regulated by protein-protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin V is overexpressed in cystic fibrosis (CF), and given the functional properties of annexin V and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. Using co-immunoprecipitation and overlay experiments, we show that annexin V is associated with nucleotide-binding domain 1 (NBD1) of CFTR. Surface plasmon resonance (SPR) indicated different KD values in the absence and presence of both calcium and ATP, suggesting that this interaction is calcium- and ATP-dependent. Using an siRNA approach and overexpression, we showed that CFTR chloride channel function and its localization in the cell membranes were dependent on annexin V expression. We concluded that annexin V is necessary for normal CFTR chloride channel activity. Furthermore, we show that CFTR and annexin V are partially co-distributed in normal epithelial cells in human bronchi. In conclusion, we show for the first time that annexin V is associated with CFTR and is involved in its function.     

Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP

The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] chloride channel is activated by cyclic nucleotide-dependent phosphorylation and ATP binding, but also by non-phosphorylation-dependent mechanisms. Other CFTR functions such as regulation of exocytotic protein secretion are also activated by cyclic nucleotide elevating agents. A soluble protein comprising the first NBD (nucleotide-binding domain) and R-domain of CFTR (NBD1-R) was synthesized to determine directly whether CFTR binds cAMP. An equilibrium radioligand-binding assay was developed, firstly to show that, as for full-length CFTR, the NBD1-R protein bound ATP. Half-maximal displacement of [3H]ATP by non-radioactive ATP at 3.5 microM and 3.1 mM was demonstrated. [3H]cAMP bound to the protein with different affinities from ATP (half-maximal displacement by cAMP at 2.6 and 167 microM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 microM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cAMP binding. Thus binding of cAMP to CFTR is consistent with a role in activation of protein secretion, a process defective in CF gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective DeltaF508-CFTR trafficking.     

Side chain and backbone contributions of Phe508 to CFTR folding

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.     

Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.     

The intact CFTR protein mediates ATPase rather than adenylate kinase activity

The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.     

ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.     

A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating

Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P¹,P⁵-di(adenosine-5′)pentaphosphate (Ap₅A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap₅A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap₅A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap₅A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap₅A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis.     

Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents by the chemical solvent tetrahydrofuran

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I(sc)) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I(sc), we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I(sc) in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K(D)) of 134 mM. This THF-induced increase in I(sc) was enhanced by forskolin (10 microM), inhibited by the PKA inhibitor H-89 (10 microM) and the thiazolidinone CFTR(inh)-172 (10 microM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I(sc) in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl(-) channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the triangle upR-S660A-CFTR Cl(-) channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.     

Nucleotide Transport Through the Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the superfamily of ATP-binding cassette (ABC) transporters, also known as traffic ATPases, which are implicated in the movement of various substrates. Recent studies indicate that CFTR and other closely related ABC transporters are also implicated in the movement of cellular ATP. This is the subject of current controversy. Therefore, evidence for the movement of cellular nucleotides by expression of CFTR and related molecules, as well as the potential significance of ATP-permeable channels in cell physiology, are reviewed in this study. The hypothesis is thus forwarded for the improper delivery of cellular ATP to the extracellular milieu by a dysfunctional CFTR, to be a relevant factor in the onset of cystic fibrosis.     

Phloxine B interacts with the cystic fibrosis transmembrane conductance regulator at multiple sites to modulate channel activity

The fluorescein derivative phloxine B is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR). Low micromolar concentrations of phloxine B stimulate CFTR Cl(-) currents, whereas higher concentrations of the drug inhibit CFTR. In this study, we investigated the mechanism of action of phloxine B. Phloxine B (1 microm) stimulated wild-type CFTR and the most common cystic fibrosis mutation, DeltaF508, by increasing the open probability of phosphorylated CFTR Cl(-) channels. At each concentration of ATP tested, the drug slowed the rate of channel closure without altering the opening rate. Based on the effects of fluorescein derivatives on transport ATPases, these data suggest that phloxine B might stimulate CFTR by binding to the ATP-binding site of the second nucleotide-binding domain (NBD2) to slow the dissociation of ATP from NBD1. Channel block by phloxine B (40 microm) was voltage-dependent, enhanced when external Cl(-) concentration was reduced and unaffected by ATP (5 mm), suggesting that phloxine B inhibits CFTR by occluding the pore. We conclude that phloxine B interacts directly with CFTR at multiple sites to modulate channel activity. It or related agents might be of value in the development of new treatments for diseases caused by the malfunction of CFTR.     

Differential function of the two nucleotide binding domains on cystic fibrosis transmembrane conductance regulator

The genetic disease cystic fibrosis is caused by defects in the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). CFTR belongs to the family of ABC transporters. In contrast to most other members of this family which transport substrates actively across a membrane, the main function of CFTR is to regulate passive flux of substrates across the plasma membrane. Chloride channel activity of CFTR is dependent on protein phosphorylation and presence of nucleoside triphosphates. From electrophysiological studies of CFTR detailed models of its regulation by phosphorylation and nucleotide interaction have evolved. These investigations provide ample evidence that ATP hydrolysis is crucial for CFTR gating. It becomes apparent that the two nucleotide binding domains on CFTR not only diverge strongly in sequence, but also in function. Based on previous models and taking into account new data from pre-steady-state experiments, a refined model for the action of nucleotides at two nucleotide binding domains was recently proposed.     

Expression of rat, renal NHE2 and NHE3 during postnatal development

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.     

Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.     

Characterization of the adenosinetriphosphatase and transport activities of purified cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMP-activated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido[alpha-(32)P]ATP-binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His(10)-tagged variant after solubilization with lysophosphatidylglycerol (1%) and diheptanoylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity [V(max) of 58 +/- 5 nmol min(-1) (mg of protein)(-1), K(M)(MgATP) of 0.15 mM]. In further work, we found that neither nucleotide binding nor ATPase activity was altered by phosphorylation (using protein kinase A) or dephosphorylation (with protein phosphatase 2B); we also observed inhibition (approximately 40%) of ATP hydrolysis by reduced glutathione but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTR-dependent transport of (125)I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [alpha-(32)P]ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.     

Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.     

CFTR directly mediates nucleotide-regulated glutathione flux

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.