Recombinant Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

Targeted quantitation of CFTR protein expression in vivo using immunoprecipitation & parallel reaction monitoring tandem mass spectrometry

Abstract Background The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a low-abundance membrane protein. The dysfunction of CFTR protein is the fundamental cause of cystic fibrosis (CF), a fatal genetic disease. In recent years, the novel messenger RNA (mRNA)-based therapy shows high potential to treat CF disease, by delivering CFTR mRNA into lung epithelial cells to generate fully functional CFTR replacement protein. To evaluate mRNA drug efficacy, a targeted quantitative proteomics method is needed to estimate the expression level of mRNA encoded CFTR protein. Methods In this paper, a method combining membrane protein extraction, immunoprecipitation (IP), and nanoLC-MS/MS for quantifying CFTR in lung tissue samples was reported for the first time. Absolute quantification was performed by constructing a standard curve by spiking recombinant human CFTR protein in mouse lung tissue matrix. Results This method was qualified, with good linearity of standard curve and lower limit of quantification of human CFTR at 1.4 pg per mg tissue. The coefficient of variation of back calculated concentration of all standards and their back-calculation errors were < 20%. The CFTR expression level in mouse lungs dosed with mRNA encapsulated lipid nanoparticle (LNP), and the endogenous level in wild type human lungs were measured successfully. Conclusions The result demonstrated high sensitivity, precision and accuracy of this proteomics method for quantifying low-abundance CFTR protein in lung tissue sample.

Potentiation of the cystic fibrosis transmembrane conductance regulator Cl- channel by ivacaftor is temperature independent.

Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability ( Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Po and temperature for F508del-CFTR was bell-shaped with a maximum Po at ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Po values of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating.

Involvement of the heterodimeric interface region of the nucleotide binding domain-2 (NBD2) in the CFTR quaternary structure and membrane stability

The cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the ATP-binding cassette (ABC) superfamily that functions as a chloride channel. The predicted structure of CFTR protein contains two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD1 and NBD2). The opening of the Cl− channel is directly linked to ATP-driven tight dimerization of CFTR's NBD1 and NBD2 domains. The presence of a heterodimeric interfaces (HI) region in NBD1 and NBD2 generated a head to tail orientation necessary for channel activity. This process was also suggested to promote important conformational changes in the associated transmembrane domains of CFTR, which may impact the CFTR plasma membrane stability. To better understand the role of the individual HI region in this process, we generated recombinant CFTR protein with suppressed HI-NBD1 and HI-NBD2. Our results indicate that HI-NBD2 deletion leads to the loss of the dimerization profile of CFTR that affect its plasma membrane stability. We conclude that, in addition to its role in Cl− transport, HI-NBD2 domain confers membrane stability of CFTR by consolidating its quaternary structure through interactions with HI-NBD1 region.

A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.

CFTR potentiators partially restore channel function to A561E-CFTR, a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del-CFTR.

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature-sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch-clamp techniques. Low temperature incubation delivered a small proportion of A561E-CFTR protein to the cell surface. Like F508del-CFTR, low temperature-rescued A561E-CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E-CFTR also exhibited thermoinstability, losing function more quickly than F508del-CFTR in cell-free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small-molecule ivacaftor, partially restored function to A561E-CFTR. Interestingly, ivacaftor restored wild-type levels of channel activity (as measured by open probability) to single A561E- and F508del-CFTR Cl(-) channels. However, it accentuated the thermoinstability of both mutants in cell-free membrane patches. Like F508del-CFTR, A561E-CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature-rescued A561E-CFTR. Transformational drug therapy for A561E-CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.

Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms.

The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4, and rT3 (all tested at 50 μM) inhibited CFTR in several ways: they strongly reduced CFTR open probability by impeding channel opening; they moderately decreased single-channel current amplitude, and they promoted transitions to subconductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 μM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to subconductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to subconductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.

A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution.

Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding sites for several candidate drugs in the disease treatment. We studied the effects of the application of 2-pyrimidin-7,8-benzoflavone (PBF), a strong potentiator of the CFTR, on the properties of recombinant and equimolar NBD1/NBD2 mixture in solution. The results indicate that the potentiator induces significant conformational changes of the NBD1/NBD2 dimer in solution. The potentiator does not modify the ATP binding constant, but reduces the ATP hydrolysis activity of the NBD1/NBD2 mixture. The intrinsic fluorescence and the guanidinium denaturation measurements indicate that the potentiator induces different conformational changes on the NBD1/NBD2 mixture in the presence and absence of ATP. It was confirmed from small-angle X-ray scattering experiments that, in absence of ATP, the NBD1/NBD2 dimer was disrupted by the potentiator, but in the presence of 2 mM ATP, the two NBDs kept dimerised, and a major change in the size and the shape of the structure was observed. We propose that these conformational changes could modify the NBDs-intracellular loop interaction in a way that would facilitate the open state of the channel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in <em>Saccharomyces cerevisiae</em>

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl(-) current (I(Cl.PKA)) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I(Cl.PKA) and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I(Cl.PKA) in a concentration- and voltage-dependent fashion (IC(50) at +100 mV=0.3 ± 1.5 μM and at -100 mV=5.1 ± 1.3 μM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I(Cl.PKA) (30 μM) was tested on other cardiac ion currents. Inward current at -120 mV, comprised predominantly of the inward-rectifier background K(+) current, I(K1), was reduced by ∼43% (n=5). Under selective recording conditions, the Na(+) current (I(Na)) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at -40 mV of ∼82%; n=8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca(2+) channel current (I(Ca,L)); fractional block at +10 mV of ∼49% and of ∼28% at -10 mV; n=11, with a ∼-3 mV shift in the voltage-dependence of I(Ca,L) activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I(Cl.PKA) channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.

Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA–regulated apical chloride channels in cortical collecting duct

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.

Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pH(i)). CFTR modulates pH(i) through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pH(i). Here, we investigate the direct effects of pH(i) on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pH(i) increased the open probability (P(o)) of wild-type CFTR, whereas alkaline pH(i) decreased P(o) and inhibited Cl(-) flow through the channel. Acidic pH(i) potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pH(i) inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pH(i) modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pH(i) dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH(i) and inhibition by alkaline pH(i), suggesting that site 2 is a critical determinant of the pH(i) sensitivity of CFTR. The effects of pH(i) also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl(-) channel senses directly pH(i). The direct regulation of CFTR by pH(i) has important implications for the regulation of epithelial ion transport.

In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors

In cystic fibrosis (CF) respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress towards gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF we examined the feasibility of using SV40-derived vectors (rSV40s) which may circumvent some of these obstacles. To accommodate the large CFTR cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We therefore tested whether “gutless” rSV40s could be packaged and were able to express a functional human CFTR cDNA. Results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein which localized to the plasma membrane and restored channel function to CFTR deficient cells. Similarly in vivo experiments delivering rSV40-CFTR to the lungs of Cftr−/− mice resulted in a reduction of the pathology associated with intra-tracheal pseudomona aeruginosa challenge. rSV40-CFTR treated mice had had less weight loss when compared to control treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the BAL. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted.

Activation of the Unfolded Protein Response by ΔF508 CFTR

Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient deltaF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3deltaF cells that express different levels of endogenous wild-type (WT) and recombinant deltaF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of deltaF508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and deltaF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate deltaF508 CFTR mRNA levels. The results indicate that overexpression of deltaF508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and deltaF508 CFTR expressed within the same cell.

Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl−currents by the chemical solvent tetrahydrofuran

The chemical solvent tetrahydrofuran (THF) increases short-circuit current (I(sc)) in renal epithelia endogenously expressing the cystic fibrosis transmembrane conductance regulator (CFTR). To understand how THF increases I(sc), we employed the Ussing chamber and patch-clamp techniques to study cells expressing recombinant human CFTR. THF increased I(sc) in Fischer rat thyroid (FRT) epithelia expressing wild-type CFTR with half-maximal effective concentration (K(D)) of 134 mM. This THF-induced increase in I(sc) was enhanced by forskolin (10 microM), inhibited by the PKA inhibitor H-89 (10 microM) and the thiazolidinone CFTR(inh)-172 (10 microM) and attenuated greatly in FRT epithelia expressing the cystic fibrosis mutants F508del- and G551D-CFTR. By contrast, THF (100 mM) was without effect on untransfected FRT epithelia, while other solvents failed to increase I(sc) in FRT epithelia expressing wild-type CFTR. In excised inside-out membrane patches, THF (100 mM) potentiated CFTR Cl(-) channels open in the presence of ATP (1 mM) alone by increasing the frequency of channel openings without altering their duration. However, following the phosphorylation of CFTR by PKA (75 nM), THF (100 mM) did not potentiate channel activity. Similar results were obtained with the triangle upR-S660A-CFTR Cl(-) channel that is not regulated by PKA-dependent phosphorylation and using 2'deoxy-ATP, which gates wild-type CFTR more effectively than ATP. Our data suggest that THF acts directly on CFTR to potentiate channel gating, but that its efficacy is weak and dependent on the phosphorylation status of CFTR.

VCP/p97 AAA-ATPase Does Not Interact with the Endogenous Wild-Type Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis. The most common mutation, DeltaF508 CFTR, is retained in the endoplasmic reticulum, retrotranslocated into the cytosol, and degraded by the proteasome. In a proteomics screen to identify DeltaF508 CFTR interacting proteins, we found that valosin-containing protein (VCP)/p97, a Type II AAA ATPase that is a component of the retrotranslocation machinery, binds DeltaF508 CFTR, and this interaction is stabilized by proteasomal inhibition. Since wild-type (WT) CFTR has been reported to be inefficiently processed during biogenesis with as much as 75% of the newly synthesized protein degraded by the proteasome, we examined the VCP interaction in Calu-3, T-84, and 16HBE, three epithelial cell lines that endogenously express WT CFTR. The results indicate that when WT CFTR processing is efficient, as demonstrated in Calu-3 cells, VCP does not interact. Interestingly, overexpression of recombinant WT CFTR in Calu-3 cells results in inefficient processing and VCP interaction, demonstrating that CFTR processing efficiency and the VCP interaction are tightly coupled. Furthermore, induction of ER stress and activation of the unfolded protein response result in inefficient processing of WT CFTR in Calu-3 cells and promote the WT CFTR-VCP interaction. The results support the hypothesis that components of the retrotranslocation machinery such as VCP do not interact with CFTR in epithelial cells that endogenously express WT CFTR, since under normal conditions the processing of the WT protein is efficient.

Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression

The unfolded protein response (UPR) is a cellular recovery mechanism activated by endoplasmic reticulum (ER) stress. The UPR is coordinated with the ER-associated degradation (ERAD) to regulate the protein load at the ER. In the present study, we tested how membrane protein biogenesis is regulated through the UPR in epithelia, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a model. Pharmacological methods such as proteasome inhibition and treatment with brefeldin A and tunicamycin were used to induce ER stress and activate the UPR as monitored by increased levels of spliced XBP1 and BiP mRNA. The results indicate that activation of the UPR is followed by a significant decrease in genomic CFTR mRNA levels without significant changes in the mRNA levels of another membrane protein, the transferrin receptor. We also tested whether overexpression of a wild-type CFTR transgene in epithelia expressing endogenous wild-type CFTR activated the UPR. Although CFTR maturation is inefficient in this setting, the UPR was not activated. However, pharmacological induction of ER stress in these cells also led to decreased endogenous CFTR mRNA levels without affecting recombinant CFTR message levels. These results demonstrate that under ER stress conditions, endogenous CFTR biogenesis is regulated by the UPR through alterations in mRNA levels and posttranslationally by ERAD, whereas recombinant CFTR expression is regulated only by ERAD.

Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein

Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein

Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.