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 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 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 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 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. 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.     

Cystic fibrosis transmembrane conductance regulator has an altered structure when its maturation is inhibited

Inefficient maturation and trafficking to the cell surface of the cystic fibrosis transmembrane conductance regulator (CFTR) is the primary cause of cystic fibrosis. CFTR protein that fails to mature accumulates as an immature core-glycosylated protein and is rapidly degraded. To determine how the structures of mature and immature CFTR are different, we compared the properties of CFTR that had been expressed in the presence or absence of the proteasome inhibitor, MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal). Transient expression of wild-type CFTR in the presence of submicromolar concentrations of MG-132 blocks maturation of the protein. We found that expression of CFTR in the presence of MG-132 trapped the protein in a trypsin-sensitive conformation. In addition, the structure of the carboxyl-terminus of immature and mature CFTR differed as histidine-tagged mature CFTR was preferentially recovered by metal-chelate chromatography. No chloride channel activity was detected when membranes containing immature CFTR were fused with planar lipid bilayers. These results show that expression of CFTR in the presence of MG-132 traps the protein in an altered conformation that may be inactive.     

Cystic fibrosis transmembrane conductance regulator-independent phagosomal acidification in macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.     

An unstable transmembrane segment in the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel with 12 membrane-spanning sequences, undergoes inefficient maturation in the endoplasmic reticulum (ER). Potentially charged residues in transmembrane segments may contribute to this defect in biogenesis. We demonstrate that transmembrane segment 6 of CFTR, which contains three basic amino acids, is extremely unstable in the lipid bilayer upon membrane insertion in vitro and in vivo. However, two distinct mechanisms counteract this anchoring deficiency: (i) the ribosome and the ER translocon co-operate to prevent transmembrane segment 6 from passing through the membrane co- translationally; and (ii) cytosolic domains of the ion channel post-translationally maintain this segment of CFTR in a membrane-spanning topology. Although these mechanisms are essential for successful completion of CFTR biogenesis, inefficiencies in their function retard the maturation of the protein. It seems possible that some of the disease-causing mutations in CFTR may reduce the efficiency of proper membrane anchoring of the protein.     

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. Received: 25 August 1997 / Accepted: 20 January 1998     

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.     

Glycosylation and the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.     

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.     

囊性纤维化跨膜电导调节体:ATP结合和水解门控Cl-通道

囊性纤维化跨膜电导调节体（cystic fibrosis transmembrane conductance regulator,CFTR）是一种Cl^-通道,属于ATP结合（ATP-binding cassette,ABC）转运体超家族。CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化（cystic fibrosis,CF）发生的主要原因。这种疾病患者各组织上皮细胞内Cl^-转运失调。目前,与CF相关的不同突变超过1400种。CFTR调节（regulatory,R）域负责调控,核苷酸结合域（nucleotide-binding domains,NBDs）NBD1和NBD2负责ATP结合和水解门控。近期研究发现CFFR的NBDs与其它ABC蛋白一样可以二聚化。二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列（signature sequence）形成ATP结合袋（ATP-binding pockets,ABPs）ABP1和ABP2。ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用。ABP2由NBD2上的WalkA和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象。有一些CFrR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路。     

Cystic fibrosis transmembrane conductance regulator chloride channel blockers:Pharmacological,biophysical and physiological relevance

Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney dis-ease. Drugs that interact directly with CFTR are there-fore of interest in the treatment of a number of diseasestates. This review focuses on one class of small mol-ecules that interacts directly with CFTR, namely inhibi-tors that act by directly blocking chloride movementthrough the open channel pore. In theory such com-pounds could be of use in the treatment of diarrheaand polycystic kidney disease, however in practice allknown substances acting by this mechanism to inhibitCFTR function lack either the potency or specificity forin vivo use. Nevertheless, this theoretical pharmaco-logical usefulness set the scene for the developmentof more potent, specific CFTR inhibitors. Biophysically,open channel blockers have proven most useful as ex-perimental probes of the structure and function of theCFTR chloride channel pore. Most importantly, the useof these blockers has been fundamental in developing afunctional model of the pore that includes a wide innervestibule that uses positively charged amino acid sidechains to attract both permeant and blocking anionsfrom the cell cytoplasm. CFTR channels are also subjectto this kind of blocking action by endogenous anionspresent in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physio-logical control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR chan-nel structure and function, and of how CFTR activity is controlled by its local environment.     

Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator

Mismanaged protein trafficking by the proteostasis network contributes to several conformational diseases, including cystic fibrosis, the most frequent lethal inherited disease in Caucasians. Proteostasis regulators, as cystamine, enable the beneficial action of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators in ΔF508-CFTR airways beyond drug washout. Here we tested the hypothesis that functional CFTR protein can sustain its own plasma membrane (PM) stability. Depletion or inhibition of wild-type CFTR present in bronchial epithelial cells reduced the availability of the small GTPase Rab5 by causing Rab5 sequestration within the detergent-insoluble protein fraction together with its accumulation in aggresomes. CFTR depletion decreased the recruitment of the Rab5 effector early endosome antigen 1 to endosomes, thus reducing the local generation of phosphatidylinositol-3-phosphate. This diverts recycling of surface proteins, including transferrin receptor and CFTR itself. Inhibiting CFTR function also resulted in its ubiquitination and interaction with SQSTM1/p62 at the PM, favoring its disposal. Addition of cystamine prevented the recycling defect of CFTR by enhancing BECN1 expression and reducing SQSTM1 accumulation. Our results unravel an unexpected link between CFTR protein and function, the latter regulating the levels of CFTR surface expression in a positive feed-forward loop, and highlight CFTR as a pivot of proteostasis in bronchial epithelial cells.