Mucus aberrant properties in CF: Insights from cells and animal models.

Cystic fibrosis (CF), an autosomal genetic disorder caused by the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, is characterized by mucus accumulation in the lungs, the intestinal tract, and the pancreatic ducts. Mucins are high-molecular-weight glycoproteins that govern the biochemical and biophysical properties of mucus. In the CF lung, increased mucus viscoelasticity is associated with decreased mucociliary clearance and defects in host defense mechanisms. The link between defective ion channel and abnormal mucus properties has been investigated in studies involving cell and animal models. In this review article, we discuss recent progress toward understanding the different regions and cells that express CFTR in the airways and how mucus is produced and cleared from the lungs. In addition, we reflect on animal models that provided insights into the organization and the role of the mucin network and how mucus and antimicrobial activities act in concert to protect the lungs from invading pathogens.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Mucus strands from submucosal glands initiate mucociliary transport of large particles.

Mucus produced by submucosal glands is a key component of respiratory mucociliary transport (MCT). When it emerges from submucosal gland ducts, mucus forms long strands on the airway surface. However, the function of those strands is uncertain. To test the hypothesis that mucus strands facilitate transport of large particles, we studied newborn pigs. In ex vivo experiments, interconnected mucus strands moved over the airway surface, attached to immobile spheres, and initiated their movement by pulling them. Stimulating submucosal gland secretion with methacholine increased the percentage of spheres that moved and shortened the delay until mucus strands began moving spheres. To disrupt mucus strands, we applied reducing agents tris-(2-carboxyethyl)phosphine and dithiothreitol. They decreased the fraction of moving spheres and delayed initiation of movement for spheres that did move. We obtained similar in vivo results with CT-based tracking of microdisks in spontaneously breathing pigs. Methacholine increased the percentage of microdisks moving and reduced the delay until they were propelled up airways. Aerosolized tris-(2-carboxyethyl)phosphine prevented those effects. Once particles started moving, reducing agents did not alter their speed either ex vivo or in vivo. These findings indicate that submucosal glands produce mucus in the form of strands and that the strands initiate movement of large particles, facilitating their removal from airways.

Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus.

Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ∼30-50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.

Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses.

Gel-forming mucins form distinct morphologic structures in airways.

Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.

Alteration of protein function by a silent polymorphism linked to tRNA abundance.

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl- transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Relationships among CFTR expression, HCO3- secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ∼10-50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl(-) secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3 (-) secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3 (-) at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR(+/-) or CFTR(+/∆F508)) expressed CFTR and secreted HCO3 (-) at ∼50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3 (-) secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl(-) secretion, the amount of CFTR is rate-limiting for HCO3 (-) secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers.

Airway acidification initiates host defense abnormalities in cystic fibrosis mice.

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.

Acidic pH increases airway surface liquid viscosity in cystic fibrosis.

Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non-CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HCO3- concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator-dependent alkalinization to abnormal CF ASL. In addition, we found that increasing Ca2+ concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing Ca2+ concentration, and/or altering electrostatic interactions in ASL might benefit early CF.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility.

RATIONALE: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. OBJECTIVES: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. METHODS: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. MEASUREMENTS AND MAIN RESULTS: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. CONCLUSIONS: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

Glycaemic regulation and insulin secretion are abnormal in cystic fibrosis pigs despite sparing of islet cell mass.

Diabetes is a common and significant co-morbidity in cystic fibrosis (CF). The pathogenesis of cystic fibrosis related diabetes (CFRD) is incompletely understood. Because exocrine pancreatic disease is similar between humans and pigs with CF, the CF pig model has the potential to contribute significantly to the understanding of CFRD pathogenesis. We determined the structure of the endocrine pancreas in fetal, newborn and older CF and non-CF pigs and assessed endocrine pancreas function by intravenous glucose tolerance test (IV-GTT). In fetal pigs, pancreatic insulin and glucagon density was similar between CF and non-CF. In newborn and older pigs, the insulin and glucagon density was unchanged between CF and non-CF per total pancreatic area, but increased per remnant lobular tissue in CF reflecting exocrine pancreatic loss. Although fasting glucose levels were not different between CF and non-CF newborns, CF newborns demonstrated impaired glucose tolerance and increased glucose area under the curve during IV-GTT. Second phase insulin secretion responsiveness was impaired in CF newborn pigs and significantly lower than that observed in non-CF newborns. Older CF pigs had elevated random blood glucose levels compared with non-CF. In summary, glycaemic abnormalities and insulin secretion defects were present in newborn CF pigs and spontaneous hyperglycaemia developed over time. Functional changes in CF pig pancreas were not associated with a decline in islet cell mass. Our results suggest that functional islet abnormalities, independent of structural islet loss, contribute to the early pathogenesis of CFRD.

Impaired mucus detachment disrupts mucociliary transport in a piglet model of cystic fibrosis.

Lung disease in people with cystic fibrosis (CF) is initiated by defective host defense that predisposes airways to bacterial infection. Advanced CF is characterized by a deficit in mucociliary transport (MCT), a process that traps and propels bacteria out of the lungs, but whether this deficit occurs first or is secondary to airway remodeling has been unclear. To assess MCT, we tracked movement of radiodense microdisks in airways of newborn piglets with CF. Cholinergic stimulation, which elicits mucus secretion, substantially reduced microdisk movement. Impaired MCT was not due to periciliary liquid depletion; rather, CF submucosal glands secreted mucus strands that remained tethered to gland ducts. Inhibiting anion secretion in non-CF airways replicated CF abnormalities. Thus, impaired MCT is a primary defect in CF, suggesting that submucosal glands and tethered mucus may be targets for early CF treatment.

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs.

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

Pancreatic and biliary secretion are both altered in cystic fibrosis pigs.

The pancreas, liver, and gallbladder are commonly involved in cystic fibrosis (CF), and acidic, dehydrated, and protein-rich secretions are characteristic findings. Pancreatic function studies in humans have been done by sampling the jejunal fluid. However, it has been difficult to separately study the function of pancreatic and biliary systems in humans with CF, because jejunal fluid contains a mixture of bile and pancreatic fluids. In contrast, pancreatic and biliary ducts open separately into the porcine intestine; therefore, biliary and pancreatic fluid can be individually analyzed in CF pigs. We studied newborn wild-type (WT) and CF pigs and found that CFTR was localized to the pancreatic ducts. We collected bile and pancreatic fluid and analyzed pancreatic enzymes with activity assays and immunoblot. Pancreatic enzyme expression was significantly decreased in CF compared with WT pigs. The volume and pH of pancreatic fluid were significantly lower and protein concentration was >5-fold higher in CF pigs. Secretin stimulation increased pancreatic fluid volume and pH in WT, but not CF, pigs. Baseline bile volume did not differ between WT and CF pigs, but volume did not increase in response to secretin in CF pigs. Bile pH was lower and protein concentration was twofold higher in CF pigs. These results indicate that pancreatic and biliary secretions are altered in CF pigs. Abnormal pancreatic and biliary secretion in CF may have important implications in disease pathogenesis.

A microRNA network regulates expression and biosynthesis of wild-type and DeltaF508 mutant cystic fibrosis transmembrane conductance regulator.

Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl(-) permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl(-) transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

Human-mouse cystic fibrosis transmembrane conductance regulator (CFTR) chimeras identify regions that partially rescue CFTR-ΔF508 processing and alter its gating defect.

The ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the most common cause of cystic fibrosis. The mutation disrupts biosynthetic processing, reduces channel opening rate, and decreases protein lifetime. In contrast to human CFTR (hCFTR)-ΔF508, mouse CFTR-ΔF508 is partially processed to the cell surface, although it exhibits a functional defect similar to hCFTR-ΔF508. To explore ΔF508 abnormalities, we generated human-mouse chimeric channels. Substituting mouse nucleotide-binding domain-1 (mNBD1) into hCFTR partially rescued the ΔF508-induced maturation defect, and substituting mouse membrane-spanning domain-2 or its intracellular loops (ICLs) into hCFTR prevented further ΔF508-induced gating defects. The protective effect of the mouse ICLs was reverted by inserting mouse NBDs. Our results indicate that the ΔF508 mutation affects maturation and gating via distinct regions of the protein; maturation of CFTR-ΔF508 depends on NBD1, and the ΔF508-induced gating defect depends on the interaction between the membrane-spanning domain-2 ICLs and the NBDs. These appear to be distinct processes, because none of the chimeras repaired both defects. This distinction was exemplified by the I539T mutation, which improved CFTR-ΔF508 processing but worsened the gating defect. Our results, together with previous studies, suggest that many different NBD1 modifications improve CFTR-ΔF508 maturation and that the effect of modifications can be additive. Thus, it might be possible to enhance processing by targeting several different regions of the domain or by targeting a network of CFTR-associated proteins. Because no one modification corrected both maturation and gating, perhaps more than a single agent will be required to correct all CFTR-ΔF508 defects.