C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.     

Organic solutes rescue the functional defect in delta F508 cystic fibrosis transmembrane conductance regulator

The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (Delta F508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of Delta F508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of Delta F508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested Delta F508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing Delta F508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-Delta F508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-Delta F508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the Delta F508 and wild type CFTR and inhibited Delta F508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of Delta F508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of Delta F508 CFTR.     

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.     

Conformational and temperature-sensitive stability defects of the delta F508 cystic fibrosis transmembrane conductance regulator in post-endoplasmic reticulum compartments

Deletion of phenylalanine at position 508 (DeltaF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that DeltaF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous DeltaF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of DeltaF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued DeltaF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 degrees C is, most likely, due to decreased conformational stability of the rescued DeltaF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native DeltaF508 CFTR.     

CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.     

Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): Slow degradation of wild-type and ΔF508 CFTR in surface membrane preparations of immortalized airway epithelial cells

The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [³⁵S]Met with chase periods of 5–24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 μM castanospermine, which is an inhibitor of processing α-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR. © 1996 Wiley-Liss, Inc.     

Role of calnexin in the ER quality control and productive folding of CFTR; differential effect of calnexin knockout on wild-type and DeltaF508 CFTR

Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.     

Complement yourself: transcomplementation rescues partially folded mutant proteins

Cystic fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing ΔF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the F508 deletion mutant. Transcomplementation rescues the processing and channel activity of ΔF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.     

Chemical rescue of deltaF508-CFTR mimics genetic repair in cystic fibrosis bronchial epithelial cells

In a previous study of sodium 4-phenylbutyrate (4-PBA)-responsive proteins in cystic fibrosis (CF) IB3-1 bronchial epithelial cells, we identified 85 differentially expressed high abundance proteins from whole cellular lysate (Singh, O. V., Vij, N., Mogayzel, P. J., Jr., Jozwik, C., Pollard, H. B., and Zeitlin, P. L. (2006) Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells. J. Proteome Res. 5, 562-571). In the present work we hypothesize that a subset of heat shock proteins that interact with cystic fibrosis transmembrane conductance regulator (CFTR) in common during chemical rescue and genetic repair will identify therapeutic networks for targeted intervention. Immunocomplexes were generated from total cellular lysates, and three subcellular fractions (endoplasmic reticulum (ER), cytosol, and plasma membrane) with anti-CFTR polyclonal antibody from CF (IB3-1), chemically rescued CF (4-PBA-treated IB3-1), and genetically repaired CF (IB3-1/S9 daughter cells repaired by gene transfer with adeno-associated virus-(wild type) CFTR). CFTR-interacting proteins were analyzed on two-dimensional gels and identified by mass spectrometry. A set of 16 proteins known to act in ER-associated degradation were regulated in common and functionally connected to the protein processing, protein folding, and inflammatory response. Some of these proteins were modulated exclusively in ER, cytosol, or plasma membrane. A subset of 4-PBA-modulated ER-associated degradation chaperones (GRP94, HSP84, GRP78, GRP75, and GRP58) was observed to associate with the immature B form of CFTR in ER. HSP70 and HSC70 interacted with the C band (mature form) of CFTR at the cell surface. We conclude that chemically rescued CFTR associates with a specific set of HSP70 family proteins that mark therapeutic interactions and can be useful to correct both ion transport and inflammatory phenotypes in CF subjects.     

Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity

Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.     

Pharmacological rescue of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) detected by use of a novel fluorescence platform

Numerous human diseases arise because of defects in protein folding, leading to their degradation in the endoplasmic reticulum. Among them is cystic fibrosis (CF), caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR ), an epithelial anion channel. The most common mutation, F508del, disrupts CFTR folding, which blocks its trafficking to the plasma membrane. We developed a fluorescence detection platform using fluorogen-activating proteins (FAPs) to directly detect FAP-CFTR trafficking to the cell surface using a cell-impermeant probe. By using this approach, we determined the efficacy of new corrector compounds, both alone and in combination, to rescue F508del-CFTR to the plasma membrane. Combinations of correctors produced additive or synergistic effects, improving the density of mutant CFTR at the cell surface up to ninefold over a single-compound treatment. The results correlated closely with assays of stimulated anion transport performed in polarized human bronchial epithelia that endogenously express F508del-CFTR. These findings indicate that the FAP-tagged constructs faithfully report mutant CFTR correction activity and that this approach should be useful as a screening assay in diseases that impair protein trafficking to the cell surface.     

Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors

Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William''s E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators.     

Calreticulin negatively regulates the cell surface expression of cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.     

Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes

To investigate the degradation mechanism of misfolded membrane proteins from the cell surface, we used mutant cystic fibrosis transmembrane conductance regulators (CFTRs) exhibiting conformational defects in post-Golgi compartments. Here, we show that the folding state of CFTR determines the post-endocytic trafficking of the channel. Although native CFTR recycled from early endosomes back to the cell surface, misfolding prevented recycling and facilitated lysosomal targeting by promoting the ubiquitination of the channel. Rescuing the folding defect or down-regulating the E1 ubiquitin (Ub)-activating enzyme stabilized the mutant CFTR without interfering with its internalization. These observations with the preferential association of mutant CFTRs with Hrs, STAM-2, TSG101, hVps25, and hVps32, components of the Ub-dependent endosomal sorting machinery, establish a functional link between Ub modification and lysosomal degradation of misfolded CFTR from the cell surface. Our data provide evidence for a novel cellular mechanism of CF pathogenesis and suggest a paradigm for the quality control of plasma membrane proteins involving the coordinated function of ubiquitination and the Ub-dependent endosomal sorting machinery.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, DeltaF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous DeltaF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous DeltaF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

Surface expression of the cystic fibrosis transmembrane conductance regulator mutant DeltaF508 is markedly upregulated by combination treatment with sodium butyrate and low temperature

The DeltaF508 gene mutation prevents delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane. The current study examines the biochemical basis for the upregulation of DeltaF508 CFTR expression by sodium butyrate and low temperature. Surface CFTR protein expression was determined by quantitative immunoblot following surface biotinylation and streptavidin extraction. CF gene expression was measured by Northern analysis and CFTR function by forskolin-stimulated (125)I efflux. Butyrate increased DeltaF508 mRNA levels and protein expression but did not increase the biochemical or functional expression of DeltaF508 CFTR at the cell surface. Low temperature increased the biochemical and functional expression of DeltaF508 CFTR at the cell surface but did not increase CFTR mRNA levels. Combining treatments led to a synergistic increase in both DeltaF508 mRNA and surface protein levels that results from the stabilization of CFTR mRNA and protein by low temperature. These findings indicate that surface expression of DeltaF508 CFTR can be markedly enhanced by carefully selected combination agents.     

Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.     

Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, ΔF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous ΔF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous ΔF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

Matrine modulates HSC70 levels and rescues ΔF508-CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl(-) channel located in the plasma membrane, and its malfunction results in cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. Most CF patients carry the deletion of Phe508 (ΔF508 mutation); this mutation prevents the delivery of the CFTR to its correct cellular location, the apical (lumen-facing) membrane of epithelial cells. Molecular chaperones play a central role in determining the fate of ΔF508-CFTR. In this report, we show that the Matrine, a quinolizidine alkaloid, downregulates the expression of the molecular chaperone HSC70 and increases the protein levels of ΔF508-CFTR in human alveolar basal epithelial cells (A549 cell line), stably transfected with a ΔF508-CFTR-expressing construct. Moreover, Matrine induced ΔF508-CFTR release from endoplasmic reticulum to cell cytosol and its localization on the cell membrane. Interestingly, downregulation of HSC70 resulted in increased levels of ΔF508-CFTR complexes with the co-chaperone BAG3 that in addition appeared to co-localize with the mutated protein on the cell surface. These results shed new light on ΔF508-CFTR interactions with proteins of the chaperones/co-chaperones system and could be useful in strategies for future medical treatments for CF.