Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl⁻) channel and a regulator of outwardly rectifying Cl⁻ channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl⁻ channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3–1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, ³⁶Cl⁻ efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl⁻ channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted α-helices 5 and 6, forms an essential part of the Cl⁻ channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl⁻ channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is found at the apical region of exocrine epithelial cells, both at the cell surface and in an apically localized intracellular compartment. To determine if this internal pool was due to endocytosis, a technique was developed that allows the rate of CFTR internalization from the cell surface to be monitored. A two-step periodate/hydrazide biotinylation procedure was used to derivatize cell surface glycoconjugates. Because both of these steps are required for derivatization and are conducted at 4 degrees C, the inclusion of a 37 degrees C incubation between the treatments resulted in an assay for the internalization of cell surface glycoconjugates. CFTR was found to be targeted to a rapidly recycling endocytic pathway, as approximately 50% of cell surface CFTR was internalized within minutes and unavailable for biotinylation. In contrast, the major glycoproteins of the apical surface were not significantly endocytosed during even longer incubations at 37 degrees C. Elevating cAMP levels either by forskolin or cAMP analogs, which has been shown to activate CFTR chloride channel activity, inhibited CFTR internalization. However, cAMP did not affect the internalization of G551D CFTR, a naturally occurring Gly-551-->Asp mutant that is expressed at the cell surface but lacks normal ion-channel function. In addition, the inhibition by cAMP of CFTR was not observed when cells were depleted of cellular chloride. The presence of CFTR in epithelial cells had previously been shown to confer a cAMP-mediated inhibition on the rate of fluid-phase endocytosis. This effect was not seen in chloride-depleted cells, suggesting that CFTR's ion-channel function and localization to incipient endosomes may be responsible for the observed inhibition. The finding that CFTR is targeted to the endocytic pathway may provide insight into the role of CFTR in normal exocrine function. In addition, these findings suggest that the expression of a regulated ion channel in a membranous subcellular compartment provides a mechanism by which a cell can regulate vesicular trafficking through that compartment.     

Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.     

Cystic fibrosis transmembrane conductance regulator modulators reduce the risk of recurrent acute pancreatitis among adult patients with pancreas sufficient cystic fibrosis

BackgroundApproximately 1 in 5 patients with pancreas sufficient cystic fibrosis (PS-CF) will develop acute pancreatitis (AP). It is not known whether ivacaftor alone or in combination with other CFTR (cystic transmembrane regulator) modulators (tezacaftor or lumacaftor) can reduce the risk of AP in patients with PS-CF and AP history.MethodsWe retrospectively queried the CF registry at our institution for adult patients with PS-CF, a documented history of AP and initiation of CFTR modulators for pulmonary indications. Patient characteristics including demographics, CFTR genotype, pancreatitis risk factors, pancreatic exocrine function and other relevant laboratory, imaging parameters were obtained from the time of the sentinel AP episode through the follow-up period.ResultsA total of 15 adult CF patients were identified with mean age of 44.1 years (SD ± 13.8). In the 24 months preceding CFTR modulator initiation, six of these patients had at least 1 episode of AP with median of 2 episodes [1.75, 2.5]. None of the patients had evidence of pancreatic calcifications or exocrine pancreas insufficiency at the time of CFTR modulator initiation. The mean duration of follow-up after CFTR modulator initiation was 36.7 months (SD ± 21.5). None of the patients who remained on CFTR modulators developed an episode of AP or required hospitalization for AP related abdominal pain during follow-up.ConclusionsCFTR modulators, alone or in combination, substantially reduce the risk of recurrent AP over a mean follow-up period of 3 years in adult patients with PS-CF and a history of prior AP. These data suggest that any augmentation of CFTR function can reduce the risk of pancreatitis.     

Revisiting cystic fibrosis transmembrane conductance regulator structure and function

The cystic fibrosis transmembrane conductance regulator (CFTR) is a channel/enzyme which mediates passive diffusion of chloride and bicarbonate through epithelial cell membranes. It is expressed in many cell types throughout the body, but in the airways it is found mainly in secretory serous cells of the submucosal glands. CFTR belongs to a large super-family of ATP binding cassette transporters that have two nucleotide binding domains with characteristic sequences or "motifs". Although most other ATP binding cassette transporters consume ATP to actively transport various substrates, in CFTR the interactions of ATP with nucleotide binding domains control opening and closing of the channel pore (i.e., channel gating). Recent high resolution structures of bacterial nucleotide binding domains combined with new biochemical and electrophysiological studies of CFTR itself have led to major advances in our understanding of CFTR gating. For example, it is now clear that the ATPase activity of CFTR is not strictly required for its channel activity. CFTR has at least two distinct gating modes; one dependent on hydrolysis and the other requiring only stable ATP binding. In this article we discuss a working hypothesis for CFTR that incorporates these recent findings and discuss some interesting implications of the paradigm shift for other aspects of CFTR function and dysfunction.     

Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (delta F508-CFTR), reduces Cl- secretion, but the fatal consequences of CF have been difficult to rationalize solely in terms of this defect. The aim of this study was to determine the role of CFTR in HCO3- transport across cell membranes. HCO3- permeability was assessed from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein] and of channel activity (patch clamp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or delta F508-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4Cl) and incubated in Na(+)-free (N-methyl-D-glucamine substitution) solutions (to block Na(+)-dependent pHi regulatory mechanisms), pHi remained acidic (pH approximately 6.5) until the cells were treated with 20 microM forskolin (increases cellular [cAMP]); pHi then increased toward (but not completely to) control level (pHi 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in delta F508 and mock-transfected cells. This Na(+)-independent, forskolin-dependent pHi recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not delta F508-CFTR or mock-transfected) cells in Cl(-)-containing, HCO3(-)-free solutions showed Cl- channels with a linear I/V relationship and a conductance of 10.4 +/- 0.5 pS in symmetrical 150 mM Cl-. When channels were incubated with different [Cl-] and [HCO3-] on the inside and outside, the Cl-/HCO3- permeability ratio (determined from reversal potentials of I/V curves) was 3.8 +/- 1.0 (mean +/- SEM; n = 9); the ratio of conductances was 3.9 +/- 0.5 (at 150 mM Cl- and 127 mM HCO3-. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMP-dependent influx of HCO3- through channels that conduct HCO3- about one-quarter as efficiently as it conducts Cl-. Under physiological conditions, the electrochemical gradients for both Cl- and HCO3- are directed outward, so CFTR likely contributes to the epithelial secretion of both ions. HCO3- secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO3- may lead to decreased pH of the luminal fluid.     

Expression of cystic fibrosis transmembrane conductance regulator in liver tissue from patients with cystic fibrosis

The authors examined the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and its relationship to histopathological changes in cystic fibrosis (CF) liver tissue. Immunohistochemistry was used to examine expression of CFTR, intercellular adhesion molecule-1 (ICAM-1) and liver cell-type markers in liver cryosections in 11 patients with CF-associated liver disease, and non-CF controls with (n = 17) and without (n = 3) liver disease. In CF patients prominent inflammatory infiltrates were not found, yet hepatic stellate cells were identified within fibrotic areas around bile ducts. Proliferating bile ducts displayed ICAM-1 immunoreactivity in 3 cases, but bile ducts were otherwise negative. In 2 patients homozygous for R764X and for 1112delT no CFTR immunoreactivity was detected. Bile-duct epithelial cells in patients carrying the DeltaF508 mutation displayed aberrant cytoplasmic immunolocalization of CFTR, as determined with confocal laser scanning microscopy, in contrast to the distinct CFTR expression at the luminal surface seen in controls. No clear relationship between CFTR expression and fibrosis or inflammation was evidenced in CF patients. In conclusion, these findings are consistent with an impairment of DeltaF508 CFTR processing in intrahepatic biliary epithelium. ICAM-1 expression on bile-duct epithelial cells and inflammatory infiltrates were rare findings in CF liver tissue, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease.     

Base treatment corrects defects due to misfolding of mutant cystic fibrosis transmembrane conductance regulator

BACKGROUND & AIMS: The most common form of the disease-causing cystic fibrosis transmembrane conductance regulator mutation, DeltaF508, leads to a misfolded protein that undergoes endoplasmic reticulum-associated degradation. Retrieval of misfolded protein from the cis-Golgi or pre-Golgi intermediate compartment is a critical factor in endoplasmic reticulum retention and degradation of DeltaF508 protein. Therefore, the inhibition of retrograde Golgi-to-endoplasmic reticulum traffic by the alkalinization of Golgi lumen may permit functional DeltaF508 protein to reach the cell surface. METHODS: Functional and biochemical effects of alkaline treatment on misfolded cystic fibrosis transmembrane conductance regulator-induced defects were measured in CFPAC-1 cells, which endogenously express DeltaF508 cystic fibrosis transmembrane conductance regulator, and in CHO cells, which heterologously express DeltaF508 cystic fibrosis transmembrane conductance regulator. The animal survival rate and the functional expression of cystic fibrosis transmembrane conductance regulator proteins were analyzed in homozygous DeltaF508 mice after chronic treatment with weak base NaHCO3. RESULTS: In CFPAC-1 and CHO cells, intracellular alkalization by reducing carbon dioxide concentrations in a carbon dioxide incubation chamber or intra-Golgi alkalization by bafilomycin A1 treatment increased the membrane expression of DeltaF508 protein and cystic fibrosis transmembrane conductance regulator-dependent anion transport. Notably, chronic administration of NaHCO3 increased the long-term survival of homozygous DeltaF508 mice and induced the functional expression of cystic fibrosis transmembrane conductance regulator in the luminal membrane of intestinal epithelium. CONCLUSIONS: We found that base treatments correct misfolded cystic fibrosis transmembrane conductance regulator-induced defects in vitro and in vivo. These results imply that the alkalization of intracellular compartments, in particular, Golgi or pre-Golgi intermediate compartments, can be a potential therapeutic target for the loss-of-function type of conformational diseases.     

Cystic fibrosis transmembrane conductance regulator gene abnormalities in patients with asthma and recurrent neutrophilic bronchitis

The present case series describes four patients with asthma, airway hyper-responsiveness and neutrophilic bronchitis who harboured abnormal cystic fibrosis transmembrance conductance regulator (CFTR) gene mutations. It serves both to alert clinicians to consider CFTR-related disease in both young and elderly patients with persistent neutrophilic bronchitis, and to highlight the potential utility of future genetic testing for CFTR abnormalities in patients with asthma and recurrent bronchitis or pansinusitis, and the role of nebulized hypertonic saline as a therapeutic option in these patients.     

Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

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-95/Discs-large/ZO-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.     

Survey of cystic fibrosis transmembrane conductance regulator genotypes in primary sclerosing cholangitis

A variety of cholestatic liver diseases appear to primarily affect the biliary epithelium, including cystic fibrosis (CF). CF results from a defect in the chloride channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of CF patients have a genomic deletion in F508, other mutations of CFTR may result in less severe clinical presentations and outcomes. Recently, CFTR has been shown to be involved in secretin-stimulated choleresis in intrahepatic bile duct epithelial cells. Cholestasis in cystic fibrosis appears to result from defective chloride transport across the biliary epithelium and is the only cholestatic disease of bile ducts for which a cellular defect has been identified. Primary sclerosing cholangitis (PSC) is a cholestatic disease with histological and cholangiographic features similar to CF. The purpose of this pilot study was to explore whether there is an increased prevalence of CFTR gene mutations in PSC. Nineteen patients with PSC were screened for 32 CFTR mutations. Two patients exhibited mutations in one allele, yielding a carrier rate of 10.6%, not statistically different from the general U.S. population carrier rate of 4%.Supported by Duke University Medical Center Small Research Grant; American Gastroenterological Association Research Award; Grant-in-Aid of Research, Indiana-Purdue University at Indianapolis; Project Development Program, Research and Sponsored Programs, Indiana University at Indianapolis; and Glaxo Institute for Digestive Health Basic Research Award.     

Detection of cystic fibrosis transmembrane conductance regulator activity in early-phase clinical trials

Advances in our understanding of cystic fibrosis pathogenesis have led to strategies directed toward treatment of underlying causes of the disease rather than treatments of disease-related symptoms. To expedite evaluation of these emerging therapies, early-phase clinical trials require extension of in vivo cystic fibrosis transmembrane conductance regulator (CFTR)-detecting assays to multicenter trial formats, including nasal potential difference and sweat chloride measurements. Both of these techniques can be used to fulfill diagnostic criteria for the disease, and can discriminate various levels of CFTR function. Full realization of these assays in multicenter clinical trials requires identification of sources of nonbiological intra- and intersite variability, and careful attention to study design and statistical analysis of study-generated data. In this review, we discuss several issues important to the performance of these assays, including efforts to identify and address aspects that can contribute to inconsistent and/or potentially erroneous results. Adjunctive means of detecting CFTR including mRNA expression, immunocytochemical localization, and other methods are also discussed. Recommendations are presented to advance our understanding of these biomarkers and to improve their capacity to predict cystic fibrosis outcomes.