Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast

Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5-10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I- uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I-V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies.     

The cystic fibrosis transmembrane conductance regulator and ATP

The purpose of this study was to monitor changes in preoperative routines following recommendations given in 1989 by the Swedish Council on Technology Assessment and by the Swedish Consensus Conference on Preoperative Routines, both suggesting a more individualized utilization of preoperative testing. This was a multicenter, prospective, repetitive study conducted at seven Swedish hospitals providing surgical care. The subjects included all patients presenting at the operating theaters for surgical interventions requiring general or regional anesthesia. The main outcome measures were the frequency of reports of performed preoperative ECG, chest x-ray, and analysis of serum concentration of potassium. Before general recommendations were issued in 1989, 47% of the patients had had a preoperative ECG, 26% had had a chest x-ray, and in 61% serum potassium concentration had been measured. In 1991 these frequencies had been reduced by 5, 6, and 9 percentage points, respectively (all figures given standardized for age and sex). Large differences were found between individual hospitals. Although recommendations suggesting a more restrictive and individualized utilization of preoperative testing have sparked important revisions in Swedish hospitals, there seems to be substantial potential remaining for further reduction of perfunctory use of preoperative screening.     

How do cystic fibrosis transmembrane conductance regulator mutations produce lung disease?

In recent years, several functions of the cystic fibrosis transmembrane conductance regulator have been discovered, yet the pathophysiology of the pulmonary disease in cystic fibrosis remains unclear. At the cellular level, functions of this protein include regulation of chloride and sodium transport at the cell membrane and in intracellular organelles, regulation of protein trafficking, and posttranslational processing of glycoconjugates. Elucidation of these functions has led to several hypotheses to account for how defects in the cystic fibrosis transmembrane conductance regulator produce pulmonary disease, but a clear understanding of the pathophysiologic links between the cellular functions of the cystic fibrosis transmembrane conductance regulator and organ dysfunction has been hampered by the lack of ideal model systems. Current evidence suggests that defects in the cystic fibrosis transmembrane conductance regulator lead to alterations in periciliary fluid homeostasis, mucus hydration, mucin secretion, and apical membrane protein structure. In turn, these alterations impair mucociliary clearance and promote bacterial infection, which then leads to chronic airway inflammation and the development of bronchiectasis.     

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

OBJECTIVE: The effect of bleaching agents on bacterial adherence to polished surfaces of composite resin restorations was assessed in vitro. STUDY DESIGN: Samples of light-curing composite resins were treated with either 10% carbamide peroxide or 10% hydrogen peroxide for 1, 3, or 7 days. Bacterial adherence of Streptococcus mutans, Streptococcus sobrinus, and Actinomyces viscosus to the treated resin samples was analyzed and compared with adherence to nonbleached controls. RESULTS: A 10% solution of carbamide peroxide caused a significant increase in surface adherence of Streptococcus mutans and Streptococcus sobrinus after 3 days (P < .01). A 10% solution of hydrogen peroxide caused a significant increase in surface adherence of Streptococcus mutans and Streptococcus sobrinus after 3 and 7 days (P < .01). A decrease in adherence of Actinomyces viscosus was found after treatment with 10% hydrogen peroxide for 7 days (P < .05). CONCLUSIONS: It appears that bleaching agents may affect adherence of certain cariogenic microorganisms to the outer surfaces of composite resin restorations.     

An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl- channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.     

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 alpha-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.     

Hormonal regulation of cardiac cystic fibrosis transmembrane conductance regulator chloride channels

The chloride conductance that arises from the stimulation of beta-adrenoceptors has been shown to be carried by a cardiac isoform of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. This brief review will focus first upon the cellular signal transduction system for the activation of this type of Cl- channels and then its regulation by catecholamines, muscarinic agonists, endothelin-1 and angiotensin-II. Both in physiological and pathological conditions, the complex interaction of these agonists modulates the Cl- conductance, which is potentially arrhythmogenic by shortening the action potential duration and inducing the depolarization.     

Topological model of membrane domain of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator is a cAMP-regulated chloride channel. We used molecular modelling to predict 3-D models for the CFTR membrane domain. Hydropathy and residue conservation in all CFTRs as well as in other proteins suggested that the membrane domain is a 12-helix bundle. If the domain is enclosing a channel for chloride, it could be made of five helices. We propose two structural models in which both lumenal and cytoplasmic entrances to the chloride pore have a ring of positively charged residues. The inner surface of the channel is covered with neutral polar plus one or two charged residues. Helices that are not directly involved in the chloride channel could organise to form a second channel; a dimeric symmetrical structure is proposed. Analysis raised interest for helix 5: this hydrophobic fragment is conserved in all CFTRs and aligns with segments present in several different ion channels and transporters. The existence of an FFXXFFXXF motif is proposed. Helix 5 could be an important domain of CFTRs. The models agree with available data from pathological mutations but does not account for the membrane insertion of a hydrophilic fragment of NBDI.     

Prediction of the antigenic sites of the cystic fibrosis transmembrane conductance regulator protein by molecular modelling

Antibodies are powerful tools for studying the in situ localization and physiology of proteins. The prediction of epitopes by molecular modelling has been used successfully for the papilloma virus, and valuable antibodies have been raised [Müller et al. (1990) J. Gen. Virol., 71, 2709-2717]. We have improved the modelling approach to allow us to predict epitopes from the primary sequences of the cystic fibrosis transmembrane conductance regulator. The procedure involves searching for fragments of primary sequences likely to make amphipathic secondary structures, which are hydrophilic enough to be at the surface of the folded protein and thus accessible to antibodies. Amphipathic helices were predicted using the methods of Berzofsky, Eisenberg and J?hnig. Their hydrophobic-hydrophilic interface was calculated and drawn, and used to predict the orientation of the helices at the surface of the native protein. Amino acids involved in turns were selected using the algorithm of Eisenberg. Tertiary structures were calculated using 'FOLDING', a software developed by R. Brasseur for the prediction of small protein structures [Brasseur (1995) J. Mol. Graphics, in press]. We selected sequences that folded as turns with at least five protruding polar residues. One important property of antibodies is selectivity. To optimize the selectivity of the raised antibodies, each sequence was screened for similarity (FASTA) to the protein sequence from several databanks. Ubiquitous sequences were discarded. This approach led to the identification of 13 potential epitopes in the cystic fibrosis transmembrane conductance regulator: seven helices and six loops.     

Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.     

Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator

The cytosolic nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (NBD1 and NBD2) mediate ATP-dependent opening and closing of the Cl- channel pore. To learn more about NBD structure and function, we introduced a cysteine residue into the Walker A motif or the LSGGQ motif of each NBD and examined modification by N-ethylmaleimide (NEM). Covalent modification of either Walker A motif partially inhibited cystic fibrosis transmembrane conductance regulator channel activity, decreasing the open state probability by prolonging the long closed duration. An increase in cytosolic ATP concentration slowed the rate of modification. The data suggest that both NBDs interact with ATP to influence channel opening and that inhibition by NEM modification was in part due to decreased ATP binding. When cysteine was placed in the NBD2 Walker A motif, it was modified more rapidly than when it was placed in NBD1, suggesting that the NBDs are not structurally or functionally identical. Modification of a cysteine inserted in the LSGGQ motif of either NBD1 or NBD2 also inhibited channel activity. The rate of modification was comparable with that of a thiol in free solution, suggesting that the LSGGQ motif resides in a surface-exposed position in both NBDs.     

Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator

The anion-selective channel CFTR (cystic fibrosis transmembrane conductance regulator), whose dysfunction is responsible for the onset of cystic fibrosis, is regulated by cAMP through the activation of protein kinase A (PKA). The nature of this activation process is unknown. In the present study, patch-clamp techniques were applied to both mouse mammary adenocarcinoma cells expressing human epithelial CFTR (CFTR cells) and cultured neonatal rat ventricular myocytes (NRVM), to determine whether CFTR is modulated by the actin cytoskeleton, and whether the actin cytoskeleton may be implicated in the cAMP-stimulated activation of the channel protein. Acute changes in the actin cytoskeleton by addition of cytochalasin D (CD) activated whole-cell currents in CFTR cells and NRVM. Addition of actin to excised, inside-out patches also activated CFTR. A functional characterization of CFTR in either cell type included cAMP-induced, linear whole-cell and single-channel currents in symmetrical Cl-, permeability to ATP, and inhibition by either diphenylamine-carboxylate (DPC) or a monoclonal antibody raised against CFTR. Incubation of CFTR cells and NRVM with CD for over 6 h prevented CFTR activation either by the cAMP pathway under whole-cell conditions or by PKA under excised inside-out conditions. Thus a complete derangement of the actin cytoskeleton prevents the cAMP-dependent activation of CFTR. CFTR activation, however, was restored by subsequent addition of actin. In summary, changes in actin filament organization modulate CFTR channel activity by a mechanism entailing a direct interaction between actin filaments and CFTR.     

Channel-lining residues in the M3 membrane-spanning segment of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride-selective channel. Residues from the 12 putative membrane-spanning segments form at least part of the channel lining. We need to identify the channel-lining residues in order to understand the structural basis for the channel's functional properties. Using the substituted-cysteine-accessibility method we mutated to cysteine, one at a time, 24 consecutive residues (Asp192-Ile215) in the M3 membrane-spanning segment. Cysteines substituted for His199, Phe200, Trp202, Ile203, Pro205, Gln207, Leu211, and Leu214 reacted with charged, sulfhydryl-specific reagents that are derivatives of methanethiosulfonate (MTS). We infer that these residues are on the water-accessible surface of the protein and probably form a portion of the channel lining. When plotted on an alpha-helical wheel the exposed residues from Gln207 to Leu214 lie within an arc of 60 degrees; the exposed residues in the cytoplasmic half (His199-Ile203) lie within an arc of 160 degrees. We infer that the secondary structures of the extracellular and cytoplasmic halves of M3 are alpha-helical and that Pro205, in the middle of the M3 segment, may bend the M3 segment, moving the cytoplasmic end of the segment in toward the central axis of the channel. The bend in the M3 segment may help to narrow the channel lumen near the cytoplasmic end. In addition, unlike full-length CFTR, the current induced by the deletion construct, Delta259, is inhibited by the MTS reagents, implying that the channel structure of Delta259 is different than the channel structure of wild-type CFTR.     

Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.     

Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in madin-darby canine kidney cells

The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl-) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl- secretion by stimulating CFTR Cl- channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patch-clamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl- secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl- secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.     

Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue-specific expression and regulation of CFTR function when animal models are used in gene therapy studies.     

Acute responses of non-human primates to airway delivery of an adenovirus vector containing the human cystic fibrosis transmembrane conductance regulator cDNA

Recombinant human adenovirus (Ad) vectors are leading candidates for human gene therapy for cystic fibrosis (CF) based on demonstration of efficient transfer of exogenous genes to rodent respiratory epithelium in vivo and human respiratory cells in vitro. The safety of Ad-mediated gene transfer to the respiratory epithelium and acute (up to 21 days) clinical responses to airway delivery of a replication-deficient recombinant, E1-, E3- Ad type 5-based vector containing the human cystic fibrosis transmembrane conductance regulator cDNA (AdCFTR) were evaluated in rhesus monkeys. Airway delivery of an Ad vector with the lacZ marker gene demonstrated beta-galactosidase expression in epithelial cells. Animals administered intratracheal AdCFTR demonstrated human CFTR cDNA expression in airway epithelial cells. Animals administered AdCFTR intranasal, and 24 hr later, intrabronchial [2 x 10(7) to 5 x 10(10) plaque-forming units (pfu), n = 12], in a fashion similar to a proposed human protocol, or only intrabronchial (10(11) pfu, n = 3), had no significant changes in clinical parameters compared to vehicle controls (n = 6). Microscopic analysis of the lung by necropsy or bronchoalveolar lavage demonstrated a dose-dependent increase in inflammatory cells, primarily lymphocytes, in the area where AdCFTR was delivered, which persisted for at least 2 months in some animals. Serum anti-Ad type 5 neutralizing antibody titers did not rise and shed Ad was not detected. The presence of AdCFTR DNA, analyzed by the polymerase chain reaction (PCR), was not detected in organs outside the lung. These data demonstrate that AdCFTR is well tolerated in non-human primates, although there is dose-dependent inflammation in the lung not clinically apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cystic fibrosis transmembrane conductance regulator as a marker of human pancreatic duct development

BACKGROUND & AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a small conductance adenosine 3',5'-cyclic monophosphate (cAMP)-activated chloride ion channel found in the apical membranes of epithelia within the pancreas, airway, intestine, bile duct, sweat gland, and male genital ducts. Pancreatic insufficiency is a feature of about 85% of patients with cystic fibrosis and is believed to be caused by pancreatic autolysis after pancreatic duct obstruction. The aim of this study was to investigate the expression of CFTR in the pancreas from early development to postnatal life to establish whether the CFTR plays a key role in development of the pancreatic duct epithelium. METHODS: Expression of CFTR from the start of the mid-trimester of human development through term to adult life by messenger RNA (mRNA) in situ hybridization was examined. RESULTS: CFTR mRNA is detected throughout the pancreatic duct epithelium and its pattern of expression follows the differentiation of the duct system. CONCLUSIONS: CFTR is a valuable marker of human pancreatic duct cell development and differentiation.