Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Activity Research Articles

Bacterial Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR)

Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR’s regulatory “R” domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.

EG-VEGF, BV8, and their receptor expression in human bronchi and their modification in cystic fibrosis: Impact of CFTR mutation (delF508).

Enhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca(2+) and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion.

Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

Cyst enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

NMR evidence for differential phosphorylation-dependent interactions in WT and ΔF508 CFTR

The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DeltaF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and DeltaF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild-type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N-terminal intracellular domain (ICD). Phosphorylation of DeltaF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full-length CFTR and expand our understanding of the molecular basis of the DeltaF508 defect.

α-Aminoazaheterocyclic-Methylglyoxal Adducts Do Not Inhibit Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Activity

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. In a recent study, several highly polar alpha-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (J Pharmacol Exp Ther 322:1023-1035, 2007), more than 10,000-fold better than that of thiazolidinone and glycine hydrazide CFTR inhibitors previously identified by high-throughput screening. In this study, we resynthesized and evaluated the alpha-aminoazaheterocyclic-methylglyoxal adducts reported to have high CFTR inhibition potency (compounds 5, 7, and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 microM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp experiments, and yellow fluorescence protein-fluorescence quenching. As positive controls, approximately 100% of CFTR inhibition was found by thiazolidinone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by alpha-aminoazaheterocyclic-methylglyoxal adducts.

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.

Annexin V is directly involved in cystic fibrosis transmembrane conductance regulator's chloride channel function

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-activated chloride channel, which is regulated by protein–protein interactions. The extent to which CFTR is regulated by these interactions remains unknown. Annexin V is overexpressed in cystic fibrosis (CF), and given the functional properties of annexin V and CFTR we considered whether they are associated and if so whether this has implications for CFTR function. Using co-immunoprecipitation and overlay experiments, we show that annexin V is associated with nucleotide-binding domain 1 (NBD1) of CFTR. Surface plasmon resonance (SPR) indicated different K D values in the absence and presence of both calcium and ATP, suggesting that this interaction is calcium- and ATP-dependent. Using an siRNA approach and overexpression, we showed that CFTR chloride channel function and its localization in the cell membranes were dependent on annexin V expression. We concluded that annexin V is necessary for normal CFTR chloride channel activity. Furthermore, we show that CFTR and annexin V are partially co-distributed in normal epithelial cells in human bronchi. In conclusion, we show for the first time that annexin V is associated with CFTR and is involved in its function.

Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

Determination of CFTR chloride channel activity and pharmacology using radiotracer flux methods

Flux studies using either radioisotopes or ion-selective electrodes are a convenient method to assay the function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel. Here, we described three different protocols to study the properties, regulation and pharmacology of the CFTR Cl − channel in populations of cells and artificial vesicles. These techniques are widely used to evaluate the function of wild-type and mutant CFTR prior to detailed analyses using the patch-clamp technique. Moreover, they have proved especially valuable in the search for new drugs to treat cystic fibrosis.

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Pharmacology of CFTR chloride channel activity.

Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109-S144, 1999. - The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl- channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl-. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

Characterization of CFTR expression and chloride channel activity in human endothelia.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a low-conductance, cAMP-regulated chloride (Cl-) channel in a variety of cell types, such as exocrine epithelial cells. Our results demonstrate that human primary endothelial cells isolated from umbilical vein (HUVEC) and lung microvasculature (HLMVEC) also express CFTR as determined via RT-PCR and immunohistochemical and immunoprecipitation analyses. Moreover, Cl- efflux and whole cell patch-clamp analyses reveal that HUVEC (n = 6 samples, P < 0.05) and HLMVEC (n = 5 samples, P < 0.05) display cyclic nucleotide-stimulated Cl- transport that is inhibited by the CFTR selective Cl- channel blocker glibenclamide but not by the blocker DIDS, indicative of CFTR Cl- channel activity. Taken together, these findings demonstrate that human endothelial cells derived from multiple organ systems express CFTR and that CFTR functions as a cyclic nucleotide-regulated Cl- channel in human endothelia.

A Conserved Region of the R Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is Important in Processing and Function

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR) connects the two halves of the protein, each of which possess a transmembrane-spanning domain and a nucleotide binding domain. Phosphorylation of serine residues, which reside mostly within the C-terminal two-thirds of the R domain, is required for nucleotide-dependent activation of CFTR chloride channel activity. The N terminus of the R domain is also likely to be important in CFTR function, since this region is highly conserved among CFTRs of different species and exhibits sequence similarity with the "linker region" of the related protein, P-glycoprotein. To date, however, the role of this region in CFTR channel function remains unknown. In this paper, we report the effects of five disease-causing mutations within the N terminus of the CFTR-R domain. All five mutants exhibit defective protein processing in mammalian HEK-293 cells, suggesting that they are mislocalized and fail to reach the cell surface. However, in the Xenopus oocyte, three mutants reached the plasma membrane. One of these mutants, L619S, exhibits no detectable function, whereas the other two, D614G and I618T, exhibit partial activity as chloride channels. Single channel analysis of these latter two mutants revealed that they possess defective rates of channel opening, consistent with the hypothesis that the N terminus of the R domain participates in ATP-dependent channel gating. These findings support recent structural models that include this region within extended boundaries of the first nucleotide binding domain.

Structural basis for specificity and potency of xanthine derivatives as activators of the CFTR chloride channel.

1. On the basis of their structure, we compared the ability of 35 xanthine derivatives to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel stably expressed in chinese hamster ovary (CHO) cells using the cell-attached patch clamp and iodide efflux techniques. 2. Activation of CFTR channels was obtained with 3-mono, 1,3-di or 1,3,7-tri-substituted alkyl xanthine derivatives (enprofylline, theophylline, aminophylline, IBMX, DPMX and pentoxifylline). By contrast, xanthine derivatives substituted at the C8- or N9-position failed to open CFTR channels. 3. The CFTR chloride channel activity was blocked by glibenclamide (100 microM) but not by DIDS (100 microM). 4. Activation of CFTR by xanthines was not mimicked by the calcium ionophore A23187, adenosine, UTP, ATP or the specific phosphodiesterase inhibitors rolipram, Ro 20-1724 and milrinone. In addition, we found no correlation between the effect of xanthines on CFTR and on the cellular cyclic AMP or ATP levels. 5. We then synthesized a series of 3,7-dimethyl-1-alkyl xanthine derivatives; among them, 3,7-dimethyl-1-propyl xanthine and 3,7-dimethyl-1-isobutyl xanthine both activated CFTR channels without increasing the intracellular cyclic AMP level, while the structurally related 3,7-dimethyl-1-(2-propenyl) xanthine and 3,7-dimethyl-1-(oxiranyl methyl) xanthine were inactive. 6. Our findings delineate a novel function for xanthine compounds and identify the molecular features that enable xanthine activation of CFTR. These results may be useful in the development of new molecules for studying the pharmacology of chloride channels.

Cystic fibrosis transmembrane conductance regulator activation stimulates endosome fusion in vivo.

Previous studies have suggested a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of intracellular vesicular trafficking. A quantitative fluorescence method was used to test the hypothesis that CFTR expression and activation affects endosome-endosome fusion in intact cells. Endosomes from CFTR-expressing and control (vector-transfected) Swiss 3T3 fibroblasts were labeled by internalization with 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene (Bodipy)-avidin, a fluid-phase marker whose fluorescence increases approximately 8-fold upon biotin binding. Cells were washed, chased, and then labeled with biotin-albumin or biotin-transferrin. The fraction of Bodipy-avidin-labeled endosomes that fused with biotin-containing endosomes (f(fusion)) was quantified by ratio imaging microfluorimetry. Endosome fusion in unstimulated CFTR-expressing cells was similar to that in control cells. However, in CFTR-expressing cells activated by forskolin, ffusion was increased by 1.30 +/- 0.18- and 2.65 +/- 0.17-fold for a 0 and 10 min chase time between avidin and biotin-albumin pulses; f(fusion) also increased (1.32 +/- 0.11-fold) when biotin-transferrin replaced biotin-albumin. The stimulation of endosome fusion was not due to differences in rates of endocytosis or endosomal acidification. Endosome fusion was not stimulated by forskolin in Cl--depleted CFTR-expressing cells, suggesting that the increase in endosome fusion is due to the CFTR chloride channel activity. These results provide evidence that CFTR is involved in the regulation of endosome fusion and, thus, a possible basis for the cellular defects associated with cystic fibrosis.

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

Gene Therapy for Cystic Fibrosis Using Cationic Liposome Mediated Gene Transfer: A Phase I Trial of Safety and Efficacy in the Nasal Airway. University of Alabama at Birmingham, Birmingham, Alabama

SCIENTIFIC ABSTRACT Cystic fibrosis (CF) is a common, lethal, inherited disease among Caucasian children and young adults. While the pathophysiology of CF includes many organ systems (e.g., gastrointestinal, reproductive, endocrine) the predominant cause of death is respiratory failure. Hyperviscous respiratory secretions and related chronic pulmonary infections lead to scarring and fibrosis of the lungs, deteriorating pulmonary function, and death, with an average CF life expectancy of approximately 29 years. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR functions, at least in part, as an epithelial chloride channel which resides in the apical membranes of exocrine gland epithelial cells. It has been suggested that the fluid and electrolyte content of normal respiratory mucus is dependent upon CFTR chloride channel activity, and that failure to secrete chloride by CF cells results in diminished hydration of mucous, sputum hyperviscosit...

Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is found at the apical region of exocrine epithelial cells, both at the cell surface and in an apically localized intracellular compartment. To determine if this internal pool was due to endocytosis, a technique was developed that allows the rate of CFTR internalization from the cell surface to be monitored. A two-step periodate/hydrazide biotinylation procedure was used to derivatize cell surface glycoconjugates. Because both of these steps are required for derivatization and are conducted at 4 degrees C, the inclusion of a 37 degrees C incubation between the treatments resulted in an assay for the internalization of cell surface glycoconjugates. CFTR was found to be targeted to a rapidly recycling endocytic pathway, as approximately 50% of cell surface CFTR was internalized within minutes and unavailable for biotinylation. In contrast, the major glycoproteins of the apical surface were not significantly endocytosed during even longer incubations at 37 degrees C. Elevating cAMP levels either by forskolin or cAMP analogs, which has been shown to activate CFTR chloride channel activity, inhibited CFTR internalization. However, cAMP did not affect the internalization of G551D CFTR, a naturally occurring Gly-551-->Asp mutant that is expressed at the cell surface but lacks normal ion-channel function. In addition, the inhibition by cAMP of CFTR was not observed when cells were depleted of cellular chloride. The presence of CFTR in epithelial cells had previously been shown to confer a cAMP-mediated inhibition on the rate of fluid-phase endocytosis. This effect was not seen in chloride-depleted cells, suggesting that CFTR's ion-channel function and localization to incipient endosomes may be responsible for the observed inhibition. The finding that CFTR is targeted to the endocytic pathway may provide insight into the role of CFTR in normal exocrine function. In addition, these findings suggest that the expression of a regulated ion channel in a membranous subcellular compartment provides a mechanism by which a cell can regulate vesicular trafficking through that compartment.

Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro.

We have generated several clones of Chinese hamster ovary, mouse epitheloid C127, and pig kidney epithelial LLCPK1 cells producing high levels of functional recombinant human cystic fibrosis transmembrane conductance regulator (CFTR). Processing of CFTR to the mature and fully glycosylated form in these cells is inefficient with only approximately 40% of all newly synthesized CFTR being converted to the mature form. Furthermore, expression of the most frequent mutant allele of the cystic fibrosis (CF) gene, the delta F508 mutant in these epithelial cells, indicated that it is biosynthetically arrested at the endoplasmic reticulum and fails to traffic to the plasma membrane. Using a combination of CFTR mutants and monoclonal antibodies, all the detectable recombinant CFTR in these cells was determined at least under the conditions used, to be present as a monomer. To demonstrate the feasibility of protein replacement therapy, we were able to effect the physical transfer of functional recombinant CFTR produced in Chinese hamster ovary cells to the plasma membranes of Ha3b fibroblasts, a cell line devoid of cAMP-stimulated chloride channels. Transfer of CFTR was mediated by the hemagglutinin viral fusion protein of influenza virus present on the Ha3b cells. Efficiency of transfer was up to 25% of the target cells, and CFTR chloride channel activity was detectable for up to 12 h post-fusion. Therefore, with the development of an appropriate formulation of fusogenic proteoliposome or virosome containing reconstituted purified CFTR, it should be feasible to introduce functional CFTR into CF-affected cells.

Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains.

Regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is unusual in that phosphorylated channels require cytosolic adenosine triphosphate (ATP) to open. The CFTR contains two regions predicted to be nucleotide-binding domains (NBDs); site-directed mutations in each NBD have now been shown to alter the relation between ATP concentration and channel activity, which indicates that ATP stimulates the channel by direct interaction with both NBDs. The two NBDs are not, however, functionally equivalent: adenosine diphosphate (ADP) competitively inhibited the channel by interacting with NBD2 but not by interacting with NBD1. Four cystic fibrosis-associated mutations in the NBDs reduced absolute chloride channel activity, and one mutation also decreased the potency with which ATP stimulates channel activity. Dysfunction of ATP-dependent stimulation through the NBDs may be the basis for defective CFTR chloride channel activity in some cystic fibrosis patients.