Cystic fibrosis with three mutations in the cystic fibrosis transmembrane conductance regulator gene

Summary Three mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were discovered in a pancreas-insufficient patient with cystic fibrosis (CF) who displayed an uncommon combination of almost normal chloride concentration in sweat tests and typical symptoms of gastrointestinal and pulmonary disease. The R553Q mutation was found on the maternal F508-CFTR gene. Codon 553 is located within a consensus motif of the ATP-binding cassette transport proteins at a less conserved position. Other members of this protein superfamily contain a glutamine instead of arginine at the homologous position, suggesting a modulating rather than disease-causing role of the R553Q mutation in CFTR. The amplification refractory mutation system did not detect the R553Q mutation in a further 65 normal, 113 F508, and 91 non-F508 CF chromosomes. The index case carried the R553X nonsense mutation on the paternal chromosome. The R553X mutation was present on a further 9 out of 86 German nonF508 CF chromosomes linked with the XV2c-KM19Mp6d9-J44-GATT haplotypes 2-2-2-1-1 and 1-1-2-1-2. The location of R553X on separate haplotypes including both alleles of the intragenic GATT repeat suggests an ancient and/or multiple origins of the R553X mutations. The association of the genotype of the CFTR mutation and the clinical phenotype was assessed for the patients carrying the related genotypes F508/F508 (n = 80), F508/R553X (n = 9) and F508-R553Q/R553X (n = 1). In compound heterozygotes, the median chloride concentration in pilocarpine iontophoresis sweat tests was significantly lower than in the F508 homozygotes (P < 0.01). The patient groups were significantly different with respect to the distributions of the centiles for height (P < 0.001) and weight (P < 0.01) as the most sensitive predictors of the course and prognosis in CF. Growth retardation was more pronounced in the compound heterozygotes.     

Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with alphabetagamma mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either alphabetagamma mENaC or alphabetagamma human ENaC (hENaC). Co-expression of CFTR and alphabetagamma mENaC or hENaC resulted in an approximately 3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an approximately 20% decrease in alpha-T663-hENaC-mediated currents, and essentially no change in alpha-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of alpha ENaC confer species specificity regarding ENaC inhibition by activated CFTR.     

Cystic fibrosis transmembrane conductance regulator in teleost fish

The gills and intestinal epithelia of teleost fish express cystic fibrosis transmembrane conductance regulator (CFTR), and utilize this low conductance anion channel in the apical membrane for ion secretion in seawater gill and in the basolateral membrane for ion absorption in freshwater gill. Similarly, in the intestine CFTR is present in the basolateral membrane for intestinal absorption and also in the apical membrane of secreting intestine. The expression of CFTR and the directed trafficking of the protein to the apical or basolateral membrane is salinity-dependent. The CFTR gene has been cloned and sequenced from several teleost species and although all the major elements in the human gene are present, including two nucleotide binding domains that are common to all ATP binding cassette (ABC) transporters, the sequences are divergent compared to shark or human. In euryhaline fish adapting to seawater, CFTR, localized immunocytochemically, redistributes slowly from a basolateral location to the apical membrane while ion secretory capacity increases. The facility with which teleosts regulate CFTR expression and activation during salinity adaptation make this system an appealing model for the expression and trafficking operation of this labile gene product.     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.

The etiology of allergic bronchopulmonary aspergillosis (ABPA) is not well understood. A clinical phenotype resembling the pulmonary disease seen in cystic fibrosis (CF) patients can occur in some individuals with ABPA. Reports of familial occurrence of ABPA and increased incidence in CF patients suggest a possible genetic basis for the disease. To test this possibility, the entire coding region of the cystic fibrosis transmembrane regulator (CFTR) gene was analyzed in 11 individuals who met strict criteria for the diagnosis of ABPA and had normal sweat electrolytes (< or = 40 mmol/liter). One patient carried two CF mutations (deltaF508/R347H), and five were found to carry one CF mutation (four deltaF508; one R117H). The frequency of the deltaF508 mutation in patients with ABPA was significantly higher than in 53 Caucasian patients with chronic bronchitis (P < .0003) and the general population (P < .003). These results suggest that CFTR plays an etiologic role in a subset of ABPA patients.     

Cystic fibrosis transmembrane conductance regulator gating requires cytosolic electrolytes

Cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis when nonfunctional, is an anion channel and a member of the ATP binding cassette superfamily. After phosphorylation, CFTR gates by binding and hydrolyzing ATP. We show that CFTR open probability (P(o)) also depends on the electrolyte concentration of the cytosol. Inside-out patches from Calu-3 cells were transiently exposed to solutions of 160 mm salt or solutions in which up to 90% of the salt was replaced by nonionic osmolytes such as sucrose. In lowered salt solutions, CFTR P(o) declined within 1 s to a stable lower value that depended on the electrolyte concentration, (K(1/2) approximately 80 mm NaCl). P(o) was rapidly restored in normal salt concentrations without regard to the electrolyte species. Reducing external electrolytes did not affect CFTR P(o). The same results were obtained when CFTR was stably phosphorylated with adenosine 5'-O-(thiotriphosphate). The decrease in P(o) resulted entirely from an increase in mean closed time. Increasing ATP levels up to 20-fold did not counteract the effect of low electrolytes. The same effect was observed for CFTR expressed in C127 cells but not for a different species of anion channel. Cytosolic electrolytes are an unsuspected, essential cofactor for CFTR gating.     

Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms

Recent studieshave demonstrated that several compounds with diverse structures canactivate wild-type cystic fibrosis transmembrane conductance regulator(CFTR) by non-receptor-mediated mechanisms. Some of these compoundshave been shown to enhance cAMP-dependent activation of F508-CFTR.This study was undertaken to compare the mechanisms by which genistein,IBMX, milrinone, 8-cyclopentyl-1,3-dipropylxanthine (CPX), thebenzimidazolone NS004, and calyculin A increase CFTR activity. Ourstudies demonstrate that, in transfected NIH-3T3 cells, maximalenhancements of forskolin-dependent F508-CFTR activity are greatestwith genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, andcalyculin A do not increase cellular cAMP. Because forskolin andcalyculin A increase in vivo phosphorylation of cAMP binding responseelement (CREB), the inability of milrinone, genistein, CPX, and NS004to increase CREB phosphorylation suggests that they do not stimulateprotein kinase A or inhibit phosphatase activity. Our data suggest thatthe mechanisms by which genistein and NS004 activate CFTR differ. Wealso demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement ofcAMP-dependent CFTR activity is not due to an increase in cellular cAMPand may involve a mechanism like that of genistein.

     

Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis.

Cystic fibrosis (CF) is an inherited disorder causing pancreatic, pulmonary, and sinus disease in children and young adults. Abnormal viscosity of mucous secretions is a hallmark of the disease, and is believed to be the result of altered electrolyte transport across epithelial cell membranes. The monogenic etiology of this disease has been apparent for more than 40 years, but the defective gene has only recently been identified. This was made possible because of a revolution in genetic technology, called positional cloning, which can pinpoint disease genes without previous knowledge of the abnormal protein product. The protein encoded by the gene defective in CF has been termed the CF transmembrane conductance regulator (CFTR) because of its postulated role in electrolyte transport. Studies investigating the normal function of CFTR and how mutations affect that function, thereby causing CF, have required the combined skills of clinicians, geneticists, molecular biologists, and physiologists. From this collaborative effort a greater understanding of the pathogenesis of this disorder is now emerging. It may soon be possible to introduce novel therapies derived from this new knowledge that will be aimed directly at the basic defect. An ever-increasing number of genes of unknown function will be identified by continuing advances in molecular genetic technology and the advent of the genome sequencing project. The experience in cystic fibrosis research may prove to be a paradigm for investigation of the function of genes isolated by positional cloning methods.     

Cystic fibrosis transmembrane conductance regulator trafficking is mediated by the COPI coat in epithelial cells

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted beta-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. Beta-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of beta-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the alpha-, beta-, and gamma-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.     

Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns

The selectivity of Ca2+ over Na+ is approximately 3.3-fold larger in cGMP-gated channels of cone photoreceptors than in those of rods when measured under saturating cGMP concentrations, where the probability of channel opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude. At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is approximately 7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of alpha and beta subunits. The maximal PCa/PNa of channels formed from alpha subunits of bovine rod channels is less than that of heteromeric channels formed from alpha and beta subunits. In addition, Ca2+ is a more effective blocker of channels formed by alpha subunits than of channels formed by alpha and beta subunits. The cGMP-dependent shift in divalent cation selectivity is a property of alphabeta channels and not of channels formed from alpha subunits alone.     

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.     

Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and deltaF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.     

Cystic fibrosis mutations lead to carboxyl-terminal fragments that highlight an early biogenesis step of the cystic fibrosis transmembrane conductance regulator

Inefficient delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to the surface of cells contributes to disease in the majority of cystic fibrosis patients. Analysis of cystic fibrosis-associated missense mutations in the first nucleotide binding domain (NBD1), including A455E, S549R, Y563N, and P574H, revealed reduced levels of mature CFTR with elevated levels of carboxyl-terminal polypeptide fragments of 105 and 90 kDa. These fragments appear early in biogenesis and degrade rapidly in four distinct cell types tested including the bronchial epithelial IB3-1 cell line. They were detected at highest levels with CFTRA455E where the 105-kDa fragment accounted for 40% of newly synthesized polypeptide but for only 20 and 7% of nascent wild type and mutant DeltaF508 proteins, respectively. The bands represent core- and unglycosylated forms of the same CFTR fragment supporting that precursor forms are correctly inserted into the membrane of the endoplasmic reticulum. Proteolytic cleavage would be predicted to occur on the cytosolic face of the endoplasmic reticulum within the NBD1-R domain segment, but pharmacological testing did not support involvement of the 26 S proteasome. The examined missense mutations in NBD1 manifest differently than the major mutant, DeltaF508, and highlight a critical conformational aspect of biogenesis of CFTR.     

Cystic fibrosis transmembrane conductance regulator-dependent up-regulation of Kir1.1 (ROMK) renal K+ channels by the epithelial sodium channel

The epithelial sodium channel (ENaC) and the secretory potassium channel (Kir1.1/ROMK) are expressed in the apical membrane of renal collecting duct principal cells where they provide the rate-limiting steps for Na(+) absorption and K(+) secretion. The cystic fibrosis transmembrane conductance regulator (CFTR) is thought to regulate the function of both ENaC and Kir1.1. We hypothesized that CFTR may provide a regulatory link between ENaC and Kir1.1. In Xenopus laevis oocytes co-expressing both ENaC and CFTR, the CFTR currents were 3-fold larger than those in oocytes expressing CFTR alone due to an increased expression of CFTR in the plasma membrane. ENaC was also able to increase Kir1.1 currents through an increase in surface expression, but only in the presence of CFTR. In the absence of CFTR, co-expression of ENaC was without effect on Kir1.1. ENaC-mediated CFTR-dependent up-regulation of Kir1.1 was reduced with a Liddle's syndrome mutant of ENaC. Furthermore, ENaC co-expressed with CFTR was without effect on the closely related K(+) channel, Kir4.1. We conclude that ENaC up-regulates Kir1.1 in a CFTR-dependent manner. CFTR may therefore provide the mechanistic link that mediates the coordinated up-regulation of Kir1.1 during the stimulation of ENaC by hormones such as aldosterone or antidiuretic hormone.     

Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity

Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.     

Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.     

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.