Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Channel Gating Regulation by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) First Cytosolic Loop

In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.     

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl⁻ and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl⁻ ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl⁻ ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl⁻ permeation, demonstrating their functional role in maximization of Cl⁻ flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl⁻. The location of these Cl⁻-attractive residues suggests that cytoplasmic Cl⁻ ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl⁻ ions from the cytoplasm.     

Multi-Ion Mechanism for Ion Permeation and Block in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

The mechanism of Cl⁻ ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl⁻ channels by binding to a site ∼40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl⁻ concentration. Increasing extracellular Cl⁻ concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl⁻ and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl⁻ is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl⁻ concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl⁻ and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl⁻ permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl⁻ (but not gluconate) ions and the dependence of channel conductance on Cl⁻ concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN⁻) ions and anomalous mole fraction behavior seen in Cl⁻/SCN⁻ mixtures.     

Interactions between Impermeant Blocking Ions in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore: Evidence for Anion-Induced Conformational Changes

It is well known that extracellular Cl⁻ ions can weaken the inhibitory effects of intracellular open channel blockers in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel pore. This effect is frequently attributed to repulsive ion-ion interactions inside the pore. However, since Cl⁻ ions are permeant in CFTR, it is also possible that extracellular Cl⁻ ions are directly competing with intracellular blocking ions for a common binding site; thus, this does not provide direct evidence for multiple, independent anion binding sites in the pore. To test for the possible through-space nature of ion-ion interactions inside the CFTR pore, we investigated the interaction between impermeant anions applied to either end of the pore. We found that inclusion of low concentrations of impermeant Pt(NO₂)₄²⁻ ions in the extracellular solution weaken the blocking effects of three different intracellular blockers [Pt(NO₂)₄²⁻, glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoic acid] without affecting their apparent voltage dependence. However, the effects of extracellular Pt(NO₂)₄²⁻ ions are too strong to be accounted for by simple competitive models of ion binding inside the pore. In addition, extracellular Fe(CN)₆³⁻ ions, which do not appear to enter the pore, also weaken the blocking effects of intracellular Pt(NO₂)₄²⁻ ions. In contrast to previous models that invoked interactions between anions bound concurrently inside the pore, we propose that Pt(NO₂)₄²⁻ and Fe(CN)₆³⁻ binding to an extracellularly accessible site outside of the channel permeation pathway alters the structure of an intracellular anion binding site, leading to weakened binding of intracellular blocking ions.     

Direct and Indirect Effects of Mutations at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO₂)₄²⁻ and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO₂)₄²⁻ ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO₂)₄²⁻. Inhibition by extracellular Pt(NO₂)₄²⁻ requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO₂)₄²⁻ is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO₂)₄²⁻ and extracellular Cl⁻ but not those between intracellular Pt(NO₂)₄²⁻ and extracellular Pt(NO₂)₄²⁻. We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS*

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of “rational” approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287–288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287–288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel.     

The CLIC1 Chloride Channel Is Regulated by the Cystic Fibrosis Transmembrane Conductance Regulator when Expressed in Xenopus Oocytes

CLIC proteins comprise a family of chloride channels whose physiological roles are uncertain. To gain further insight into possible means of CLIC1 channel activity regulation, this protein was expressed in Xenopus oocytes alone or in combination with the cystic fibrosis transmembrane conductance regulator (CFTR). Whole-cell currents were determined using two-electrode voltage-clamp methods. Expression of CLIC1 alone did not increase whole-cell conductance either at rest or in response to increased intracellular cyclic adenosine monophosphate (cAMP). However, expression of CLIC1 with CFTR led to increased cAMP-activated whole-cell currents compared to expression from the same amount of CFTR mRNA alone. IAA-94 is a drug known to inhibit CLIC family channels but not CFTR. In oocytes expressing both CLIC1 and CFTR, a fraction of the cAMP-activated whole-cell current was sensitive to IAA-94, whereas in oocytes expressing CFTR alone, the cAMP-stimulated current was resistant to the drug. Cell fractionation studies revealed that the presence of CFTR conferred cAMP-stimulated redistribution of a fraction of CLIC1 from a soluble to a membrane-associated form. We conclude that when expressed in Xenopus oocytes CFTR confers cAMP regulation to CLIC1 activity in the plasma membrane and that at least part of this regulation is due to recruitment of CLIC1 from the cytoplasm to the membrane.     

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to <40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.In this method, we provide comparison of the purification of CFTR in dodecyl-β-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1''-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.     

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

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 protein^1-3. 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 described^6-9 .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.     

基于蛋白质剪接的BHK细胞Ser~(660)前断裂的CFTR基因转移

利用内含肽(intein)的蛋白质反式剪接技术,研究双载体真核细胞转囊性纤维化跨膜电导调节体(CFTR)基因,通过翻译后连接成为完整的功能性CFTR蛋白.应用基因重组技术,将人CFTRcDNA于剪接反应所需保守残基Ser660前断裂为N端和C端两部分,分别与split Ssp DnaB intein编码序列融合,构建到真核表达载体pEGFP-N1和pEYFP-N1.用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),48h后Western印迹观察CFTR蛋白质的连接,并用全细胞和单通道膜片钳技术记录Cl-通道电流.基因共转染细胞可观察到明显的由蛋白质反式剪接形成的完整CFTR蛋白,膜片钳记录到较高的全细胞Cl-电流和与转野生型CFTR基因细胞相似的单Cl-通道开放活性,提示CFTR功能的恢复.内含肽可作为一种技术策略用于双载体转CFTR基因,为应用双腺相关病毒载体(AAV)转基因的囊性纤维化疾病(CF)基因治疗提供了依据.     

Cystic Fibrosis Transmembrane Conductance Regulator Is Found Within Brain Ventricular Epithelium and Choroid Plexus

Abstract: The cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator (CFTR), functions as a CI⁻ channel that is regulated by cyclic AMP-dependent phosphorylation. We have investigated the expression of CFTR protein in the rodent brain by both western blotting of samples prepared by microdissection and immunohistochemistry. CFTR was found to be expressed in choroid plexus and ependyma. In tissue sections, CFTR-like immunoreactivity was concentrated in fine puncta localized about 1–2 µm from the CSF-contacting side of ependyma and choroid plexus. CFTR in choroid plexus may play a role in the regulation of the composition of CSF by cyclic AMP-elevating agents, but the role of this chloride transporter in ependymal function remains to be determined.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Confers Glibenclamide Sensitivity to Outwardly Rectifying Chloride Channel (ORCC) in Hi-5 Insect Cells

Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, ΔF508-CFTR or E. coliβ galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 ± 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 ± 2.5 pS at −80 mV; 55 ± 4.1 pS at +80 mV) and properties. In the presence of 500 μm SITS, channel open probability (P _o ) of ORCC was reversibly reduced to 0.05 ± 0.01 in CFTR-cells, to 0.07 ± 0.02 in non-CFTR expressing cells and to 0.05 ± 0.02 in ΔF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 μm), whereas 500 μm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or ΔF508, glibenclamide dose dependently (IC₅₀= 17 μm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 μm glibenclamide reversibly reduced P _o from 0.88 ± 0.03 to 0.09 ± 0.02 (wash: P _o = 0.85 ± 0.1) in CFTR cells and from 0.89 ± 0.05 to 0.08 ± 0.05 (wash: P _o = 0.87 ± 0.1) in ΔF508 cells. In non-CFTR expressing cells, glibenclamide (100 μm) was without effect on P _o (control: P _o = 0.89 ± 0.09, glib.: P _o = 0.86 ± 0.02; wash: P _o = 0.87 ± 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells. Received: 23 October 1998/Revised: 29 December 1998     

Folding and Rescue of a Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Mutant Identified Using Human-Murine Chimeric Proteins

Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys⁵⁸⁴ interacts with residue Leu⁵⁸¹ in human CFTR Glu⁵⁸⁴ interacts with Phe⁵⁸¹ in mouse CFTR. Introduction of the murine residue (Phe⁵⁸¹) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.     

Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr^+/+ and cftr^−/− mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Allelic Variants Relate to Shifts in Faecal Microbiota of Cystic Fibrosis Patients

Introduction

In this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age.

Methods

Thirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure.

Results

Patients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced.

Conclusions

This is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a ‘systemic disease’, linking the lung and the gut in a joined axis.     

Cystic Fibrosis Transmembrane Conductance Regulator Interacts with Multiple Immunoglobulin Domains of Filamin A

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa·partner complexes as templates, we generated in silico models of IgFLNa·CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated (∼160 nm) strands, whereas CFTR is compact (6∼8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa·partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.     

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ΔF508. Previously we found that the ATP analog N⁶-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ∼7-fold. Here we show that 2′-deoxy-ATP (dATP), but not 3′-deoxy-ATP, increases the activity of G551D-CFTR by ∼8-fold. We custom synthesized N⁶-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ± 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ΔF508-CFTR by increasing its activity by 19.5 ± 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ΔF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable.