Additional Disruption of the ClC-2 Cl- Channel Does Not Exacerbate the Cystic Fibrosis Phenotype of Cystic Fibrosis Transmembrane Conductance Regulator Mouse Models* (original) (raw)
Related papers
The Journal of biological chemistry, 1992
The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of...
Induction of expression of the cystic fibrosis transmembrane conductance regulator
Journal of Biological Chemistry
The cystic fibrosis transmembrane conductance regulator (CFTR) was studied in HT-29 human colonic carcinoma cells with the aim of assessing possible mechanisms of up-regulation of its expression. CFTR was identified and quantified in total cell extracts by Western immunoblots using a monoclonal anti-CFTR antibody and was functionally assessed by tracer C1efflux from intact cells. It was found that various stimuli that lead to a sustained (28 h) elevation of intracellular cyclic AMP elicited a marked and specific increase in CFTR expression in cell membranes and concomitant activation of C1secretion. Further activation of C1secretion was obtained by additional short term activation by cyclic AMP analogues or cyclic AMPinducing agents. Blockers of transcription or translation largely depressed the CAMP-mediated induction of CFTR levels and associated function, indicating that the inductive phenomenon was at the transcriptional level. The results imply the involvement of putative cyclic AMP responsive (and related) elements that are present in the CFTR gene promoter and that are known to modulate eukaryotic gene expression. Activation of these elements by various stimuli might provide pharmacological tools for up-regulation of CFTR expression at both biochemical and physiological levels. Cystic fibrosis (CF)' or mucoviscidosis is caused by mutations in the CFTR (cystic fibrosis conductance transmembrane regulator) gene (1, 2) that result in abnormally low CAMP-activatable apical chloride conductance and consequently in a elevated potential difference across epithelial cells (1). These abnormalities are apparently responsible for the pathophysiology of epithelial tissues in the respiratory tract, pancreas, and elsewhere (1). In CFTR gene-transfected cells (3-7), the short term cAMP activation property of C1channels was shown to be conferred by CFTR and is believed to be mediated by protein kinase A (3). Recent studies provided evidence that recombinant CFTR might function in transfected cells as a CAMP-regulated channel (3, 6) whose * This work was supported in part by the Fund for Basic Research (Israel Academy of Sciences) (to W. B.), National Institutes of Health Grant HL 40158 (to Z. I. C.), and the United States-Israel Binational Science Foundation (to Z. I. C.
Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Cl- Channel by Its R Domain
Journal of Biological Chemistry, 2001
Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated C1-channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial C1-secretion. Previous studies have shown that CAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR C1-channels. We asked whether other kinases would phosphorylate CFTR in uitro and activate CFTR C1-channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca2+-independent and Ca2+dependent isoforms of protein kinase C (PKC) activated the CFTR C1-channel. Consistent with this finding, PKC also phosphorylated CFTR in uitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR C1channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in uitro. However, cGMP failed to increase the apical membrane C1permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR C1channels. These results suggest that if cGK phosphorylates CFTR in uiuo, it does so at sites not involved in CFTR C1-channel activation. Because CAMP-dependent activation of CFTR C1-channels and C1-secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR C1channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR C1-channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in uitro. Our data indicate that CFTR C1-channels can be phosphorylated and activated by PKA as well as by Caz+-dependent and Ca2+
The Biogenesis, Traffic, and Function of the Cystic Fibrosis Transmembrane Conductance Regulator
International Review of Cytology, 1997
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMPactivated chloride channel that is encoded by the gene that is defective in cystic fibrosis. This ion channel resides at the luminal surfaces and in endosomes of epithelial cells that line the airways, intestine, and a variety of exocrine glands. In this article we discuss current hypotheses regarding how CFTR functions as a regulated ion channel and how CF mutations lead to disease. We also evaluate the emerging notion that CFTR is a multifunctional protein that is capable of regulating epithelial physiology at several levels, including the modulation of other ion channels and the regulation of intracellular membrane traffic. Elucidating the various functions of CFTR should contribute to our understanding of the pathology in cystic fibrosis, the most common lethal genetic disorder among Caucasians. Protein processing.
Proceedings of the …, 1998
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 and͞or 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, 36 Cl ؊ eff lux 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 ␣-helices 5 and 6, forms an essential part of the Cl ؊ channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1͞R 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. CFTR is a transmembrane protein involved in the regulation of several processes, including the activation of outwardly rectifying Cl Ϫ channels (1, 2) and the inhibition of Na ϩ channels by cAMP-dependent protein kinase A (PKA) (3-5). Mutations in CFTR cause cystic fibrosis (CF). Both channels lose this pattern of PKA sensitivity when CFTR is absent or its function is severely compromised in mutant forms. Other members of the ATP-binding cassette (ABC) transporter superfamily also regulate other processes. For example, the multidrug transporter, MDR, may regulate volume-activated chloride channels (6-9). The sulfonylurea receptor (SUR) binds sulfonylurea compounds such as glybenclamide and confers sulfonylurea inhibition upon a separate ATP-gated K ϩ channel protein in pancreatic  cells (10-15). More recent results suggest that CFTR can act as a SUR for ATP-gated K ϩ channels in kidney (16). We have shown previously that CFTR regulates outwardly rectifying Cl Ϫ channels (ORCCs) by an autocrine mechanism involving ATP release that is CFTR dependent. The ATP released binds to purinergic receptors to stimulate ORCCs (17, 18). The mechanism of how ATP is released, either through CFTR itself or by a separate mechanism, remains highly controversial (19-21). Two possibilities are that CFTR either transports ATP directly or activates an alternate ATP-release pathway. A key question in CF research is: How does CFTR allow protein kinase A to activate separate populations of ORCCs and inhibit a distinct family of Na ϩ-conductive channels? In this study, we tested the hypothesis that the complex, multidomain structure of CFTR supports its multifunctional behavior and that separate domains within the CFTR protein perform Cl Ϫ channel function independent of its regulatory functions. We show that the ability of CFTR to regulate ORCCs is not dependent upon CFTR's Cl Ϫ channel function and that conductance regulation is separate from CFTR's ability to conduct Cl Ϫ. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proceedings of the National Academy of Sciences, 1995
Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as ...
Journal of Biological Chemistry, 1994
Cystic fibrosis results from mutations in the gene encoding the CFTR C1channel. Although CFTR occurs as an integral component of the plasma membrane, recent studies implicate CFTR in endocytic recycling and suggest that the protein may also exist in intracellular vesicular compartments. To test this, we analyzed CFTR in clathrin-coated vesicles (CCV) purified from cells constitutively expressing CFTR at high levels. CFTR immunoreactivity was detected in CCV by immunoblot and was identified as CFTR based on labeling of immunoprecipitates with protein kinase A and by tryptic phosphopeptide mapping. Fusion of uncoated CCV with planar lipid bilayers resulted in the incorporation of kinase-and ATP-activated C1-channel activity (7.8 pS at 20 "C; 11.9 pS at 37 "C), with a linear current-voltage relation under symmetrical conditions. Thus, functional CFTR occurs in CCV. Moreover, CFTR interacts with the plasma membrane specific adaptor complex during endocytosis through clathrin-coated pits. Therefore, the abundance of CFTR in the plasma membrane may be regulated by exocytic insertion and endocytic recycling, and these processes may provide an augmentation to protein kinase A activation as a mechanism for regulating CFTR C1 channels in the plasma membrane. * This work was supported in part by Cystic Fibrosis Foundation Grants F232, F270, R464, and 236, National Institutes of Health Grants DK45970, DK42017, and DK40701, and the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrincoated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae. Calu-3 cells; caveolin; endocytosis; ion channel CYSTIC FIBROSIS (CF) is the most frequently occurring recessive genetic disorder of Caucasian populations, affecting 1 in 2,500 live births. The CF gene encodes an integral membrane glycoprotein, the CF transmembrane conductance regulator (CFTR) (34), which functions as a chloride channel within the apical membrane of polarized epithelial cells (1, 2, 19). Alterations in the gene encoding CFTR give rise to a characteristic phenotype in CF cells, namely, impaired transepithelial chloride secretion in response to activation of the cAMP-mediated second messenger cascade (23, 38).
Proceedings of the National Academy of Sciences, 1998
Cystic fibrosis (CF) is a lethal inherited disease that results from abnormal chloride conduction in epithelial tissues. ClC-2 chloride channels are expressed in epithelia affected by CF and may provide a key ''alternative'' target for pharmacotherapy of this disease. To explore this possibility, the expression level of ClC-2 channels was genetically manipulated in airway epithelial cells derived from a cystic fibrosis patient (IB3-1).