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At least 19 recordsLinked to original sources
PubMed2000

Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel expressed in luminal membranes of secretory and reabsorptive epithelia. CFTR plays a predominant role in both cAMP- and Ca2+-activated secretion of electrolytes. Although Ca2+-dependent Cl- channels exist independent of CFTR in the airway epithelium, their physiological significance remains to be determined. However, CFTR seems to be the only relevant Cl- conductance in the colonic epithelium. Apart from its secretory function, CFTR also has a task in regulating the reabsorption of electrolytes by controlling the activity of the epithelial Na+ channel, ENaC. Accordingly, defects in CFTR causing the disease cystic fibrosis (CF) lead to disturbances of both the secretion and absorption of electrolytes. Therefore, it is unclear what is pathophysiologically more important for the development of CF lung disease, the impaired secretion of Cl- or the enhanced reabsorption of Na+ and consecutive hyperabsorption of electrolytes. The mechanisms of how CFTR and ENaC interact are unknown. Previous work has given rise to several interesting working hypothesis, such as direct protein interaction or interaction via cytoskeletal proteins. Recent studies demonstrate the importance of the first nucleotide binding fold of CFTR, not only for the inhibition of ENaC but also for the interaction with other ion channels. Further studies are required to demonstrate whether regulation of other ion channels and membrane transport by CFTR occur by a common mechanism.

Animals↗
PubMed1993

Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cytosolic nucleotides and by cAMP-dependent phosphorylation. In excised membrane patches, CFTR Cl- channel activity requires hydrolyzable nucleotides and Mg2+, and is inhibited by ADP. We examined the interactions between CFTR and nucleotides using 8-azidoadenosine 5'-triphosphate (8-N3-ATP), a photoactivatable ATP analog. Because CFTR functions as a membrane ion channel, we studied CFTR in membranes of Sf9 insect cells. We found that [alpha-32P]8-N3ATP specifically photolabeled CFTR, with half-maximal labeling at 10 microM 8-N3ATP in the presence of Mg2+ and 100 microM in the absence of Mg2+. The 8-N3ATP also substituted for ATP in activating CFTR Cl- channels, indicating that it interacts with the active site(s). Both ATP and GTP prevented photolabeling with half-maximal inhibition at 1 mM. ADP and adenyl-5'-yl imidodiphosphate (AMP-PNP) prevented photolabeling but at much higher concentrations, whereas AMP did not inhibit photolabeling at concentrations of up to 100 mM. Phosphorylation of CFTR was not a prerequisite for nucleotide binding. These results demonstrate that CFTR interacts directly with nucleotides at concentrations that regulate CFTR Cl- channel activity.

Adenine Nucleotides↗
PubMed2003

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Adenosine Diphosphate↗
PubMed2005

State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET+. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.

Adenosine Triphosphate↗
PubMed2006

Calreticulin negatively regulates the cell surface expression of cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.

Adenoviridae↗
PubMed1996

Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia.

Adenosine Triphosphate↗
PubMed1997

A novel model for the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most frequent mutation is the deletion of F508 in the first nucleotide binding fold (NBF1). It induces a perturbation in the folding of NBF1, which impedes posttranslational maturation of CFTR. Determination of the three-dimensional structure of NBF1 would help to understand this defect. We present a novel model for NBF1 built from the crystal structure of bovine mitochondrial F1-ATPase protein. This model gives a reasonable interpretation of the effect of mutations on the maturation of the protein and, in agreement with the CD data, leads to reconsideration of the limits of NBF1 within CFTR.

Amino Acid Sequence↗
PubMed2002

Domain-domain associations in cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-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)- and glutathione-S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic digestion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.

Amino Acid Sequence↗
PubMed1991

In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The principal manifestations of CF include increased concentration of Cl- in exocrine gland secretions, pancreatic insufficiency, chronic lung disease, intestinal blockage and malabsorption of fat, and male and female infertility. Insight into the function of CFTR can be gained by correlating its cell-specific expression with the physiology of those cells and with CF pathology. Determination of CFTR messenger RNA in rat tissues by in situ hybridization shows that it is specifically expressed in the ductal cells of the pancreas and the salivary glands. In the intestine, decreasing gradients of expression of the CFTR gene are observed on both the crypt-villus and the proximal-distal axes. This expression is consistent with CFTR being responsible for bidirectional Cl- transport, secretion in the intestinal crypts and reabsorption in the silivary gland ducts, and suggests that in these tissues CFTR functions as a regulated Cl- channel. In the lung, a broad band of hybridization includes the mucosa and submucosa of the bronchi and bronchioles. In the testis, CFTR expression is regulated during the cycle of the seminiferous epithelium. Postmeiotic expression is maximal in the round spermatids of stages VII and VIII, suggesting that CFTR plays a critical role in spermatogenesis and that deficiency of this function contributes to CF male infertility.

Animals↗
PubMed1992

Immunocytochemical analysis reveals differences between the subcellular localization of normal and delta Phe508 recombinant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation responsible for CF is the deletion of amino acid residue Phe508, with an average allelic frequency of 70%. We have isolated an anti-CFTR monoclonal antibody which specifically recognizes recombinant normal and delta Phe508-CFTR produced by a vaccinia virus expression system. Immunocytochemical analysis of L cells expressing either normal or delta Phe508-CFTR showed a marked difference in subcellular distribution. Normal CFTR had a distinct localization in the perinuclear area and was also associated with the plasma membrane. delta Phe508-CFTR essentially lacked the membrane-associated distribution and was present throughout the cytoplasm. This heterologous expression system thus provides a model system for studying the subcellular localization of different mutant forms of CFTR.

Animals↗
PubMed2000

Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by inherited mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in epithelial tissues. Most mutations in CF patients result in rapid intracellular degradation of the CFTR protein. While this defect is thought to result from abnormal protein folding, it is unclear how mutant and wild-type (WT) proteins differ in structure, how the cell is able to distinguish these differences, and how the fate of the mutant protein is determined. By examining the initial steps of CFTR assembly into the endoplasmic reticulum (ER) membrane, it has recently been shown that CFTR utilizes two redundant translocation pathways to direct N-terminus folding events. Mutations that block one pathway therefore do not alter transmembrane topology, but rather appear to disrupt intracellular trafficking through perturbations in higher order tertiary structure. These studies suggest that cellular quality control machinery acts at least in part, by monitoring proper interactions between CFTR subdomains. The end result of this process is the conversion of misfolded CFTR into a membrane-bound, polyubiquitinated complex. This complex recruits cytosolic degradation machinery to the endoplasmic reticulum membrane where CFTR is degraded as it is extracted from the lipid bilayer. Understanding how cellular machinery mediates this process will be an important step in designing strategies to modify protein folding and degradation in CF and related ion channelopathies.

Animals↗
PubMed1995

Use of a membrane potential-sensitive probe to assess biological expression of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is caused by defects in a chloride-transporting protein termed cystic fibrosis transmembrane conductance regulator (CFTR). This study presents an innovative procedure to evaluate expression of functional CFTR. The technique uses the potential-sensitive probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol or DiSBAC2(3), by single-cell fluorescence imaging. The DiSBAC2(3) method was first validated on the mouse mammary tumor cell line C127, stably expressing wild-type CFTR. Activation of protein kinase A by the cAMP-permeable analogue 8-Br-cAMP induced cell membrane depolarization consistent with expression of wild-type CFTR. The DiSBAC2(3) method is quick, simple, and reproducible, and does not require invasive cell loading procedures. The system was then applied to the cell model of the human lung tumor cell line A549, in which exogenous CFTR was expressed by infecting with the replication-deficient recombinant adenovirus AdCFTR. DiSBAC2(3) was able to detect the fraction of cells in which the expression of CFTR protein was confirmed by immunocytochemistry. The DiSBAC2(3) probe was also used in human nasal respiratory cells cultured in vitro, in which it efficiently discriminated between endogenous CFTR in normal and CF cells. Functional evaluation of CFTR function by the described method can be a useful tool to detect the expression of the CF gene transferred by adenoviral vectors for use in gene therapy trials.

Adenoviridae↗
PubMed1998

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described.

Adult↗
PubMed1997

8-cyclopentyl-1,3-dipropylxanthine and other xanthines differentially bind to the wild-type and delta F508 first nucleotide binding fold (NBF-1) domains of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Certain alkyl xanthines such as CPX (8-cyclopentyl-1,3-dipropylxanthine) stimulate Cl- efflux from cells bearing the delta F508 genotype common to most cases of cystic fibrosis. We have hypothesized that the CFTR molecule itself might be the site for CPX action, perhaps in the region of the first nucleotide binding fold (NBF-1) domain. Therefore, to test this hypothesis directly we have used a rapid membrane filtration assay to measure the kinetics of association and dissociation of [3H]CPX to both recombinant NBF-1 and recombinant NBF-1 bearing the delta F508 mutation. We report that [3H]CPX binds with higher affinity to the delta F508-NBF-1 of CFTR (Kd = 1.0 nM) than to the wild-type NBF-1 of CFTR (Kd = 17.0 nM). These Kd values were calculated from direct measurements of the association and dissociation rate constants. The rate constants for the dissociation reaction of the wild-type NBF-1 and delta F508-NBF-1 of CFTR were not different from each other. However, the corresponding rate constants for the association reaction were k(+1) (NBF-1) = 4.7 +/- 0.9 x 10(4) M(-1) s(-1) and k(+1) (delta F508-NBF-1) = 1.6 +/- 0.3 x 10(5) M(-1) s(-1), respectively. These Kd values were corroborated by equilibrium-binding experiments, which gave very similar values. We have also measured the relative displacement of various xanthines from both wild-type NBF-1 and delta F508-NBF-1, in anticipation that the order of potencies for binding might parallel the action of the different xanthines on CF cells. For wild-type NBF-1, the rank order was DA-CPX > DAX > CPX > caffeine > adenosine >> IBMX > 2-thioCPX. For delta F508-NBF-1, the rank order was DAX > CPX > caffeine > DA-CPX > adenosine >> IBMX > 2-thioCPX. These relative potencies show close parallels with previously observed relative potencies of these drugs on CF cells, and thus lend strong support to the hypothesis that the mechanism of action on CF cells may involve direct interaction with the CFTR molecule itself.

Adenosine↗
PubMed2001

Cl(-)-dependent HCO3- transport by cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) affects the function of multiple organs. The inability to maintain luminal hydration of ducts leads to their plugging and destruction of the affected organs. An exacerbating problem is the acidic pH of the fluid produced by CF patients' secretory glands. This is best documented for pancreatic secretion. Alkaline fluid secretion requires vectorial transport of electrolytes and of HCO(3)(-). The mechanism of HCO(3)(-) secretion by cystic fibrosis transmembrane conductance regulator (CFTR) expressing cells is not well understood. In the present communication we discuss results suggesting that CFTR itself can transport large amounts of HCO(3)(-) and that HCO(3)(-) transport by CFTR is mediated by a coupled, Cl(-)-dependent process that is different from a simple HCO(3)(-) conductance.

Chloride-Bicarbonate Antiporters↗
PubMed2002

Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is characterized by chronic inflammation and an imbalance in the concentrations of alveolar and lung oxidants and antioxidants, which result in cell damage. Modifications in lung glutathione concentrations are recognized as a salient feature of inflammatory lung diseases such as cystic fibrosis, and glutathione plays a major role in protection against oxidative stress and is important in modulation of apoptosis. The cystic fibrosis transmembrane conductance regulator (CFTR) is permeable to Cl(-), larger organic ions, and reduced and oxidized forms of glutathione, and the DeltaF508 CFTR mutation found in cystic fibrosis patients has been correlated with impaired glutathione transport in cystic fibrosis airway epithelia. Because intracellular glutathione protects against oxidative stress-induced apoptosis, we studied the susceptibility of epithelial cells (HeLa and IB3-1) expressing normal and mutant CFTR to apoptosis triggered by H(2)O(2). We find that cells with normal CFTR are more sensitive to oxidative stress-induced apoptosis than cells expressing defective CFTR. In addition, sensitivity to apoptosis could be correlated with glutathione levels, because depletion of intracellular glutathione results in higher levels of apoptosis, and glutathione levels decreased faster in cells expressing normal CFTR than in cells with defective CFTR during incubation with H(2)O(2). The pro-apoptotic BCL-2 family member, BAX, is also activated faster in cells expressing normal CFTR than in those with mutant CFTR under these conditions, and artificial glutathione depletion increases the extent of BAX activation. These results suggest that glutathione-dependent BAX activation in cells with normal CFTR represents an early step in oxidative stress-induced apoptosis of these cells.

Amino Acid Substitution↗
PubMed1997

Inhibition of epithelial Na+ currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is characterized by an impaired cyclic adenosine 3,5-monophosphate (cAMP) activated Cl- conductance in parallel with an enhanced amiloride sensitive Na+ conductance (ENaC) of the respiratory epithelium. Very recently, acute downregulation of ENaC by the cystic fibrosis transmembrane conductance regulator (CFTR) was demonstrated in several studies. The mechanism, however, by which CFTR exerts its inhibitory effect on ENaC remains obscure. We demonstrate that cytosolic domains of human CFTR are sufficient to induce inhibition of rat epithelial Na+ currents (rENaC) when coexpressed in Xenopus oocytes and stimulated with 3-isobutyl-1-methylxanthine (IBMX). Moreover, mutations of CFTR, which occur in cystic fibrosis, abolish CFTR-dependent downregulation of rENaC. Yeast two hybrid analysis of CFTR domains and rENaC subunits suggest direct interaction between the proteins. Enhanced Na+ transport as found in the airways of cystic fibrosis patients is probably due to a lack of CFTR dependent downregulation of ENaC.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Structure and function of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).

Chloride Channels↗