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ORKAMBI, a combination of the corrector, lumacaftor, and the potentiator, ivacaftor, partially rescues the defective processing and anion channel activity conferred by the major cystic fibrosis–causing mutation, F508del, in in vitro studies. Clinically, the improvement in lung function after ORKAMBI treatment is modest and variable, prompting the search for complementary interventions. As our previous work identified a positive effect of arginine-dependent nitric oxide signaling on residual F508del-Cftr function in murine intestinal epithelium, we were prompted to determine whether strategies aimed at increasing arginine would enhance F508del–cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in patient-derived airway epithelia. Now, we show that the addition of arginine together with inhibition of intracellular arginase activity increased cytosolic nitric oxide and enhanced the rescue effect of ORKAMBI on F508del-CFTR–mediated chloride conductance at the cell surface of patient-derived bronchial and nasal epithelial cultures. Interestingly, arginine addition plus arginase inhibition also enhanced ORKAMBI-mediated increases in ciliary beat frequency and mucociliary movement, two in vitro CF phenotypes that are downstream of the channel defect. This work suggests that strategies to manipulate the arginine–nitric oxide pathway in combination with CFTR modulators may lead to improved clinical outcomes.SIGNIFICANCE STATEMENTThese proof-of-concept studies highlight the potential to boost the response to cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, lumacaftor and ivacaftor, in patient-derived airway tissues expressing the major CF-causing mutant, F508del-CFTR, by enhancing other regulatory pathways. In this case, we observed enhancement of pharmacologically rescued F508del-CFTR by arginine-dependent, nitric oxide signaling through inhibition of endogenous arginase activity.

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 μM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR.

Cholangiocyte organoids can be used to model liver biliary disease; however, both a defined matrix to emulate cholangiocyte self‐assembly and the mechano‐transduction pathways involved therein remain elusive. A series of defined viscoelastic hyaluronan hydrogels to culture primary cholangiocytes are designed and it is found that by mimicking the stress relaxation rate of liver tissue, cholangiocyte organoid growth can be induced and expression of Yes‐associated protein (YAP) target genes could be significantly increased. Strikingly, inhibition of matrix metalloproteinases (MMPs) does not significantly affect organoid growth in 3D culture, suggesting that mechanical remodeling of the viscoelastic microenvironment—and not MMP‐mediated degradation—is the key to cholangiocyte organoid growth. By immobilizing Jagged1 to the hyaluronan, stress relaxing hydrogel, self‐assembled bile duct structures form in organoid culture, indicating the synergistic effects of Notch signaling and viscoelasticity. By uncovering critical roles of hydrogel viscoelasticity, YAP signaling, and Notch activation, cholangiocyte organogenesis is controlled, thereby paving the way for their use in disease modeling and/or transplantation. In this article, engineered hyaluronan hydrogel elucidates cell–matrix interactions and bile duct morphogenesis. The hydrogel viscoelasticity is systematically varied to promote primary cholangiocyte organoid growth from encapsulated single cells, which is mediated by Yes‐associated protein signaling. The immobilized Jagged1 in the hydrogel activates Notch signaling in 3D cultured cholangiocytes, which leads to unprecedented bile duct‐like morphogenesis.

Mechanotransduction In article 2200880by Molly S. Shoichet and co‐workers, an engineered hyaluronan hydrogel elucidates cell–matrix interactions and bile duct morphogenesis. The hydrogel viscoelasticity is systematically varied to promote primary cholangiocyte organoid growth from encapsulated single cells, which is mediated by Yes‐associated protein signaling. The immobilized Jagged1 in the hydrogel activates Notch signaling in 3D cultured cholangiocytes, which leads to unprecedented bile duct‐like morphogenesis.

There are nearly 2000 mutations in the CFTRgene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTRgene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential–sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their “theratype” or most promising targeted therapy.

Purpose:The purpose of this study was to determine the molecular consequences of the variant c.3700 A>G in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a variant that has been predicted to cause a missense mutation in the CFTR protein (p.Ile1234Val).Methods:Clinical assays of CFTR function were performed, and genomic DNA from patients homozygous for c.3700 A>G and their family members was sequenced. Total RNA was extracted from epithelial cells of the patients, transcribed into complementary DNA, and sequenced. CFTR complementary DNA clones containing the missense mutation p.Ile1234Val or a truncated exon 19 (p.Ile1234_Arg1239del) were constructed and heterologously expressed to test CFTR protein synthesis and processing.Results:In vivo functional measurements revealed that the individuals homozygous for the variant c.3700 A>G exhibited defective CFTR function. We show that this mutation in exon 19 activates a cryptic donor splice site 18?bp upstream of the original donor splice site, resulting in deletion of six amino acids (r.3700_3717del; p.Ile1234_Arg1239del). This deletion, similar to p.Phe508del, causes a primary defect in folding and processing. Importantly, Lumacaftor (VX-809), currently in clinical trial for cystic fibrosis patients with the major cystic fibrosis–causing mutation, p.Phe508del, partially ameliorated the processing defect caused by p.Ile1234_Arg1239del.Conclusion:These studies highlight the need to verify molecular and clinical consequences of CFTR variants to define possible therapeutic strategies.Genet Med 16 8, 625–632.Genetics in Medicine (2014); 16 8, 625–632. doi:10.1038/gim.2014.4

These are exciting times with the appearance of small molecule compounds in clinical trials which target the basic defects caused by mutation in the CFTR gene. This progress was enabled by years of basic research probing the molecular and cellular consequences caused by mutation and the development of methods by which to study the primary anion transport defect in a high-throughput manner by robotics. Future progress with the development of new, more effective corrector compounds is needed. Such discovery will require further progress in defining the molecular targets for effective intervention using a multidisciplinary approach, merging computational, molecular, proteomic and cell biological methods. There is also an urgent need to develop means to link the right therapeutic compound to the right patients given the heterogeneity of the CF patient population. We envision a time when mid to high-throughput methods will be married with stem cell biology to enable testing a compendium of compounds on cells derived from each individual patient. Given the rate of progress in this field- this scenario may exist in the not too distant future.

Mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) cause CF (cystic fibrosis), a fatal genetic disease commonly leading to airway obstruction with recurrent airway inflammation and infection. Pulmonary obstruction in CF has been linked to the loss of CFTR function as a regulated Cl- channel on the lumen-facing membrane of the epithelium lining the airways. We have learned much about the molecular basis for nucleotide- and phosphorylation-dependent regulation of channel activity of the normal (wild-type) version of the CFTR protein through electrophysiological studies. The major CF-causing mutation, F508del-CFTR, causes the protein to misfold and be retained in the ER (endoplasmic reticulum). Importantly, recent studies in cell culture have shown that retention in the ER can be ‘corrected’ through the application of certain small-molecule modulators and, once at the surface, the altered channel function of the major mutant can be ‘potentiated’, pharmacologically. Importantly, two such small molecules, a ‘corrector’ (VX-809) and a ‘potentiator’ (VX-770) compound are undergoing clinical trial for the treatment of CF. In this chapter, we describe recent discoveries regarding the wild-type CFTR and F508del-CFTR protein, in the context of molecular models based on X-ray structures of prokaryotic ABC (ATP-binding cassette) proteins. Finally, we discuss the promise of small-molecule modulators to probe the relationship between structure and function in the wild-type protein, the molecular defects caused by the most common mutation and the structural changes required to correct these defects.

The deletion of Phe-508 (F508del) constitutes the most prevalent cystic fibrosis-causing mutation. This mutation leads to cystic fibrosis transmembrane conductance regulator (CFTR) misfolding and retention in the endoplasmic reticulum and altered channel activity in mammalian cells. This folding defect can however be partially overcome by growing cells expressing this mutant protein at low (27°C) temperature. Chemical "correctors" have been identified that are also effective in rescuing the biosynthetic defect in F508del-CFTR, thereby permitting its functional expression at the cell surface. The mechanism of action of chemical correctors remains unclear, but it has been suggested that certain correctors [including 4-cyclohexyloxy-2-(1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl)-quinazoline (VRT-325)] may act to promote trafficking by interacting directly with the mutant protein. To test this hypothesis, we assessed the effect of VRT-325 addition on the channel activity of F508del-CFTR after its surface expression had been "rescued" by low temperature. It is noteworthy that short-term pretreatment with VRT-325 [but not with an inactive analog, 4-hydroxy-2-(1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl)-quinazoline (VRT-186)], caused a modest but significant inhibition of cAMP-mediated halide flux. Furthermore, VRT-325 decreased the apparent ATP affinity of purified and reconstituted F508del-CFTR in our ATPase activity assay, an effect that may account for the decrease in channel activity by temperature-rescued F508del-CFTR. These findings suggest that biosynthetic rescue mediated by VRT-325 may be conferred (at least in part) by direct modification of the structure of the mutant protein, leading to a decrease in its ATP-dependent conformational dynamics. Therefore, the challenge for therapy discovery will be the design of small molecules that bind to promote biosynthetic maturation of the major mutant without compromising its activity in vivo.

The deletion of Phe-508 (ΔPhe508) constitutes the most prevalent of a number of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that cause cystic fibrosis (CF). This mutation leads to CFTR misfolding and retention in the endoplasmic reticulum, as well as impaired channel activity. The biosynthetic defect can be partially overcome by small-molecule "correctors"; once at the cell surface, small-molecule "potentiators" enhance the channel activity of ΔPhe508-CFTR. Certain compounds, such as VRT-532, exhibit both corrector and potentiator functions. In the current studies, we confirmed that the inherent chloride channel activity of ΔPhe508-CFTR (after biosynthetic rescue) is potentiated in studies of intact cells and membrane vesicles. It is noteworthy that we showed that the ATPase activity of the purified and reconstituted mutant protein is directly modulated by binding of VRT-532 [4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol] ATP turnover by reconstituted ΔPhe508-CFTR is decreased by VRT-532 treatment, an effect that may account for the increase in channel open time induced by this compound. To determine whether the modification of ΔPhe508-CFTR function caused by direct VRT-532 binding is associated with structural changes, we evaluated the effect of VRT-532 binding on the protease susceptibility of the major mutant. We found that binding of VRT-532 to ΔPhe508-CFTR led to a minor but significant decrease in the trypsin susceptibility of the full-length mutant protein and a fragment encompassing the second half of the protein. These findings suggest that direct binding of this small molecule induces and/or stabilizes a structure that promotes the channel open state and may underlie its efficacy as a corrector of ΔPhe508-CFTR.

We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes — based on the structure of ivacaftor — to covalently modify the CFTR protein at the region that constitutes the drug binding site (s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry.

Mutations in ClC-5 (chloride channel 5), a member of the ClC family of chloride ion channels and antiporters, have been linked to Dent's disease, a renal disease associated with proteinuria. Several of the disease-causing mutations are premature stop mutations which lead to truncation of the C-terminus, pointing to the functional significance of this region. The C-terminus of ClC-5, like that of other eukaryotic ClC proteins, is cytoplasmic and contains a pair of CBS (cystathionine β-synthase) domains connected by an intervening sequence. The presence of CBS domains implies a regulatory role for nucleotide interaction based on studies of other unrelated proteins bearing these domains [Ignoul and Eggermont (2005) Am. J. Physiol. Cell Physiol. 289, C1369–C1378; Scott, Hawley, Green, Anis, Stewart, Scullion, Norman and Hardie (2004) J. Clin. Invest. 113, 274–284]. However, to date, there has been no direct biochemical or biophysical evidence to support nucleotide interaction with ClC-5. In the present study, we have expressed and purified milligram quantities of the isolated C-terminus of ClC-5 (CIC-5 Ct). CD studies show that the protein is compact, with predominantly α-helical structure. We determined, using radiolabelled ATP, that this nucleotide binds the folded protein with low affinity, in the millimolar range, and that this interaction can be competed with 1 μM AMP. CD studies show that binding of these nucleotides causes no significant change in secondary structure, consistent with a model wherein these nucleotides bind to a preformed site. However, both nucleotides induce an increase in thermal stability of ClC-5 Ct, supporting the suggestion that both nucleotides interact with and modify the biophysical properties of this protein.

The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. An understanding of the structure–function relationships of these proteins is essential for defining the molecular mechanisms underlying pathogenesis. To date, the X-ray crystal structures of prokaryotic ClC transporter proteins have been used to model the membrane domains of eukaryotic ClC channel-forming proteins. Clearly, the fidelity of these models must be evaluated empirically. In the present study, biochemical tools were used to define the membrane domain boundaries of the eukaryotic protein, ClC-2, a chloride channel mutated in cases of idiopathic epilepsy. The membrane domain boundaries of purified ClC-2 and accessible cysteine residues were determined after its functional reconstitution into proteoliposomes, labelling using a thiol reagent and proteolytic digestion. Subsequently, the lipid-embedded and soluble fragments generated by trypsin-mediated proteolysis were studied by MS and coverage of approx. 71% of the full-length protein was determined. Analysis of these results revealed that the membrane-delimited boundaries of the N- and C-termini of ClC-2 and the position of several extramembrane loops determined by these methods are largely similar to those predicted on the basis of the prokaryotic protein [ecClC (Escherichia coli ClC)] structures. These studies provide direct biochemical evidence supporting the relevance of the prokaryotic ClC protein structures towards understanding the structure of mammalian ClC channel-forming proteins.

Structural information is required to define the molecular basis for chloride conduction through CFTR (cystic fibrosis transmembrane conductance regulator). Towards this goal, we expressed MSD2, the second of the two MSDs (membrane-spanning domains) of CFTR, encompassing residues 857–1158 in Sf9 cells using the baculovirus system. In Sf9 plasma membranes, MSD2 migrates as expected for a dimer in non-dissociative PAGE, and confers the appearance of an anion permeation pathway suggesting that dimeric MSD2 mediates anion flux. To assess directly the function and quaternary structure of MSD2, we purified it from Sf9 cells by virtue of its polyhistidine tag and nickel affinity. Reconstitution of MSD2 into liposomes conferred a 4,4′-di-isothiocyanostilbene-2,2′-disulphonate-inhibitable, chloride-selective electrodiffusion pathway. Further, this activity is probably mediated directly by MSD2 as reaction of its single cysteine residue (Cys866) with the thiol modifying reagent, Nα(3-maleimidylpropionyl)biocytin, inhibited chloride flux. Only MSD2 dimers were labelled by Nα(3-maleimidylpropionyl)biocytin, supporting the idea that only dimeric MSD2 can mediate anion flux. As a further test of this hypothesis, we conducted a second purification procedure, wherein purified dimeric and monomeric MSD2 proteins were reconstituted separately. Only proteoliposomes containing stable MSD2 dimers mediated chloride electrodiffusion, providing direct evidence that dimeric MSD2 mediates chloride channel function. In summary, we have shown that the second membrane domain of CFTR can be purified and functionally reconstituted as a chloride channel, providing a tool for probing the structural basis of chloride conduction through CFTR.

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700–10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

Cystic fibrosis (CF) causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to mislocalization of CFTR protein from the brush border membrane of epithelial tissues and/or its dysfunction as a chloride channel. In initial reports, it was proposed that certain channels from the ClC family of chloride channels may provide compensatory or alternative pathways for epithelial chloride secretion in tissues from cystic fibrosis patients. In the present work, we provide the first evidence that ClC-4 protein is functionally expressed on the surface of the intestinal epithelium and hence, is appropriately localized to act as a therapeutic target in this CF-affected tissue. We show using confocal and electron microscopy that ClC-4 co-localizes with CFTR in the brush border membrane of the epithelium lining intestinal crypts in mouse and human tissues. In Caco-2 cells, a cell line thought to model human enterocytes, ClC-4 protein is expressed on the cell surface and also partially co-localizes with EEA1 and transferrin, marker molecules of early and recycling endosomes, respectively. Hence, like CFTR, ClC-4 may cycle between the plasma membrane and endosomal compartment. Furthermore, we show that ClC-4 functions as a chloride channel on the surface of these epithelial cells as antisense ClC-4 cDNA expression reduced the amplitude of endogenous chloride currents by 50%. These studies provide the first evidence that ClC-4 is endogenously expressed and may be functional in the brush border membrane of enterocytes and hence should be considered as a candidate channel to provide an alternative pathway for chloride secretion in the gastrointestinal tract of CF patients.

Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl−) flux in affected epithelial tissues. CFTR is a Cl−channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl−permeates. Mutations of residues lining the channel pore (e.g.R347D) are typically thought to cause disease by altering the interaction of Cl−with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in the in vivoregulation of CFTR in health and disease.

It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.

The chloride channel ClC-2has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.

The variability in intestinal disease severity in patients with cystic fibrosis (CF) has been associated with the expression of secondary modifier genes. The locus containing these modifier genes in CF patients is syntenic with a modifier locus previously associated with survival in CF transmembrane conductance regulator–knockout mice. These previous studies showed that the proportion of CF mice that survive weaning (mildly affected mice) versusthose that succumb to obstruction of the small intestine (severely affected) is related to their genetic background and the expression of modifier genes. In the present work, we show that the basal transepithelial chloride transport measured across jejuna obtained from mice of mixed genetic backgrounds segregates into two groups, some mice having low and others having high, near normal chloride transport. Further, we report that the segregation of mice with respect to intestinal chloride transport correlates with their predicted segregation on the basis of genotype at the “modifier locus.” These findings support the hypothesis that intestinal disease modification in CF mice correlates with improved chloride transport through non-CF transmembrane conductance regulator chloride channels.

The variability in intestinal disease severity in patients with cystic fibrosis (CF) has been associated with the expression of secondary modifier genes. The locus containing these modifier genes in CF patients is syntenic with a modifier locus previously associated with survival in CF transmembrane conductance regulator–knockout mice. These previous studies showed that the proportion of CF mice that survive weaning (mildly affected mice) versus those that succumb to obstruction of the small intestine (severely affected) is related to their genetic background and the expression of modifier genes. In the present work, we show that the basal transepithelial chloride transport measured across jejuna obtained from mice of mixed genetic backgrounds segregates into two groups, some mice having low and others having high, near normal chloride transport. Further, we report that the segregation of mice with respect to intestinal chloride transport correlates with their predicted segregation on the basis of genotype at the “modifier locus.” These findings support the hypothesis that intestinal disease modification in CF mice correlates with improved chloride transport through non-CF transmembrane conductance regulator chloride channels.

Assessment of the quaternary structure of membrane proteins by PAGE has been problematic owing to their relatively poor solubility in non-dissociative detergents. Here we report that several membrane proteins can be readily solubilized in their native quaternary structure with the use of the detergent perfluoro-octanoic acid (PFO). Further, PFO can be used with PAGE, thereby providing a novel, accessible tool with which to assess the molecular mass of homo-multimeric protein complexes.

TheCftr(Cystic Fibrosis Transmembrane Conductance Regulator) gene codes for an epithelial chloride (C1) channel essential for fluid secretion into the respiratory and gastrointestinal tract and from exocrine glands. Mice lacking CFTR function due to a disruption ofCftrexon 10 or exon 1 (Cftrm1UNC/m1UNCorCftrm1HSC/m1HSCmice, respectively) generally suffer from severe gastrointestinal disease resulting in death shortly after birth or at the time of weaning. However, a subgroup of theCftrm1HSC/m1HSCmice have been characterized which exhibit relatively mild intestinal pathology resulting in a noncompromised lifespan compared to the more severely affectedCftrm1UNC/m1UNCmice. We compared the ion transport capacity of the intestinal mucosa of the mildly and severely affected CF mice using thein vivotechnique of rectal potential difference (PD) measurement and found that the net calcium-activated chloride conductance toward the lumen was much greater in the rectum of mildly affected mice than in the severely affected mice. Hence, the milder phenotype may be related to the expression of a factor which enhances the net calcium-activated chloride conductance into the lumen of the intestinal tract.

Mice that have been made deficient for the cystic fibrosis transmembrane conductance regulator (Cftr) usually die of intestinal obstruction. We have created Cftr-deficient mice and demonstrate prolonged survival among backcross and intercross progeny with different inbred strains, suggesting that modulation of disease severity is genetically determined. A genome scan showed that the major modifier locus maps near the centromere of mouse chromosome 7. Electrophysiological studies on mice with prolonged survival show that the partial rectification of Cl−and Na+ion transport abnormalities can be explained in part by up-regulation of a calcium-activated Cl−conductance. Identification of modifier genes in our Cftr m1HSC/Cftr m1HSCmice should provide important insight into the heterogeneous disease presentation observed among CF patients.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Ch−channel. In most mammalian cells, the functional consequences of the most common CF mutation, ΔF508-CFTR, cannot be assessed as the mutant protein undergoes biosynthetic arrest. However, function can be studied in the baculovirus-insect cell expression system where ΔF508-CFTR does not appear to undergo such arrest. Our results show that phosphorylation-regulated Cl−channel activity of ΔF508-CFTR is similar to that of wild-type CFTR. This observation was confirmed in comparative studies of purified ΔF508-CFTR and CFTR reconstituted in planar lipid bilayers. Therefore, we suggest that this Common mutation does not result in a significant alteration in CFTR function

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

A chloride-selective channel has been found using patch-clamp electrophysiology in human skin fibroblasts and it exhibits many of the biophysical properties of the Cl −channel found in airway epithelia. As in the case of epithelial Cl −channels, Cl −channels in fibroblasts are activated at depolarized membrane potentials in excised patches, rectifying in an outward direction with a unit conductance of 33 pS at 0 mV. Furthermore, the agonists forskolin and prostaglandin E 2evoke Cl −channel activity in cell-attached patches. The effect of these agonists can be mimicked by direct application of catalytic subunit of protein kinase A with ATP and Mg 2+to the internal membrane surface of excised, inside-out patches. The Cl −channel is also sensitive to inhibition by the stilbene derivative, DIDS. These results indicate that fibroblasts may provide a convenient and available model for the study of epithelial Cl −channel regulation and accelerate efforts to determine the regulatory defect expressed in cystic fibrosis.

We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, α-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al.(Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R.(1992) Cell68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[α-32P]ATP with half-maximal binding at 25 and 50 μM, respectively. These values compare well with those reported for 8-N3-[α-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.

This report describes a novel, single-step strategy for the purification of the cystic fibrosis transmembrane conductance regulator from Sf9 cells, which will facilitate studies of the structure–function relationships of this clinically important molecule. The new method combines the use of the novel detergent sodium pentadecafluoro-octanoate with metal-affinity chromatography to produce a high yield of purified protein which can be functionally reconstituted as a chloride channel and an ATPase.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel situated on the apical membrane of epithelial cells. Our recent studies of purified, reconstituted CFTR revealed that it also functions as an ATPase and that there may be coupling between ATP hydrolysis and channel gating. Both the ATP turnover rate and channel gating are slow, in the range of 0.2 to 1 s−1, and both activities are suppressed in a disease-causing mutation situated in a putative nucleotide binding motif. Our future studies using purified protein will be directed toward understanding the structural basis and mechanism for coupling between hydrolysis and channel function.

Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl−channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl−channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.

ABSTRACTCystic Fibrosis (CF) is caused by mutations in the CF gene that lead, for the most part, to mislocalization of the protein product, the cystic fibrosis transmembrane conductance regulatory (CFTR). CFTR is a chloride channel normally situated in the apical membrane of epithelial cells where it contributes to transepithelial ion transport. In this study we demonstrated the feasibility of in vivotransfer of purified CFTR protein via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR knockout mice. Membrane incorporation of immunogold-labeled CFTR could be visualized by electron microscopy and correction of CF-related defects in ion transport measured by nasal potential difference (PD) measurements in about one-third of the animals treated. Although these initial results are promising, effectiveness of this therapeutic approach appears to be limited by the inefficient incorporation of CFTR into the apical epithelial cell membrane.

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

A chloride‐selective channel has been found using patch‐clamp electrophysiology in human skin fibroblasts and it exhibits many of the biophysical properties of the Cl−channel found in airway epithelia. As in the case of epithelial Cl−channels, Cl−channels in fibroblasts are activated at depolarized membrane potentials in excised patches, rectifying in an outward direction with a unit conductance of 33 pS at 0 mV. Furthermore, the agonists forskolin and prostaglandin E2evoke Cl−channel activity in cell‐attached patches. The effect of these agonists can be mimicked by direct application of catalytic subunit of protein kinase A with ATP and Mg2+to the internal membrane surface of excised, inside‐out patches. The Cl−channel is also sensitive to inhibition by the stilbene derivative, DIDS. These results indicate that fibroblasts may provide a convenient and available model for the study of epithelial Cl−channel regulation and accelerate efforts to determine the regulatory defect expressed in cystic fibrosis.

Tubular biological structures consisting of extracellular matrix (ECM) proteins and cells are basic functional units of all organs in animals and humans. ECM protein solutions at low concentrations (5–10 milligrams per milliliter) are abundantly used in 3D cell culture. However, their poor “printability” and minute‐long gelation time have made the direct extrusion of tubular structures in bioprinting applications challenging. Here, this limitation is overcome and the continuous, template‐free conversion of low‐concentration collagen, elastin, and fibrinogen solutions into tubular structures of tailored size and radial, circumferential and axial organization is demonstrated. The approach is enabled by a microfabricated printhead for the consistent circumferential distribution of ECM protein solutions and lends itself to scalable manufacture. The attached confinement accommodates minute‐long residence times for pH, temperature, light, ionic and enzymatic gelation. Chip hosted ECM tubular structures are amenable to perfusion with aqueous solutions and air, and cyclic stretching. Predictive collapse and reopening in a crossed‐tube configuration promote all‐ECM valves and pumps. Tissue level function is demonstrated by factors secreted from cells embedded within the tube wall, as well as endothelial or epithelial barriers lining the lumen. The described approaches are anticipated to find applications in ECM‐based organ‐on‐chip and biohybrid structures, hydraulic actuators, and soft machines. Tubular biological structures consisting of proteins and cells are basic functional units of all organs in animals and humans. An approach is presented that allows the direct extrusion of protein‐based tubular biomaterials and tissues. The approach is anticipated to find applications in bioprinted organ‐on‐chip and biohybrid structures, hydraulic actuators, and soft machines.

Cellular uptake of neutral amino acids via Na+ cotransporters is known to be associated with an increased membrane K+ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K+ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K+-selective channels are opened by Ca2+ acting from the inside of the membrane at a concentration as low as 0.1 µM.

ABSTRACTCystic fibrosis (CF) is caused by mutations in the CFTRgene and is associated with progressive and ultimately fatal infectious lung disease. There can be considerable variability in disease severity among individuals with the same CFTRmutations, and recent genome-wide association studies have identified secondary genetic factors that contribute to this. One of these modifier genes is SLC6A14, which encodes an amino acid transporter. Importantly, variants of this gene have been associated with age at first acquisition of Pseudomonas aeruginosa. In this study, we aimed to determine the function of SLC6A14 in airway epithelia and how it might affect colonization by P. aeruginosa. We show that SLC6A14is expressed in respiratory epithelial cells and transports l-arginine out of the airway surface liquid (ASL). Exposure of airway epithelia to flagellin from P. aeruginosaled to upregulation of SLC6A14expression and increased SLC6A14-dependent uptake of l-arginine from the ASL. In support of the hypothesis that l-arginine affects P. aeruginosaattachment, we showed that l-arginine supplementation promoted P. aeruginosaattachment to an abiotic surface in a dose-dependent manner. In a coculture model, we found that inhibition of SLC6A14-dependent l-arginine transport enhanced P. aeruginosaattachment. In Slc6a14−/y(knockout) mice, P. aeruginosaattachment to lung tissue was also significantly enhanced. Together, these findings suggest that SLC6A14 activity plays a role in the modification of the initial stages of airway infection by altering the level of l-arginine in the ASL, which in turn affects the attachment of P. aeruginosa.IMPORTANCECF patients with shared CFTRgene mutations show significant variability in their clinical presentation of infectious lung disease. Genome-wide association studies have been used to identify secondary genetic factors that may explain the variable susceptibility to infection by opportunistic pathogens, including P. aeruginosa, the leading cause of pathogen-induced lung damage in nonpediatric CF patients. Once identified and characterized, these secondary genetic modifiers may allow for the development of personalized medicine for patients and ultimately the extension of life. In this study, we interrogated the biological role of one of these modifiers, SLC6A14, and showed that it contributes to host defense by depleting extracellular arginine (an attachment-promoting metabolite for P. aeruginosa) from the airway surface liquid.

ABSTRACTWzx flippases are crucial for bacterial cell surface polysaccharide assembly as they transport undecaprenyl pyrophosphate-linked sugar repeat units from the cytoplasmic to the periplasmic leaflets of the inner membrane (IM) for final assembly. Our recently reported three-dimensional (3D) model structure of Wzx from Pseudomonas aeruginosaPAO1 (WzxPa) displayed a cationic internal vestibule and functionally essential acidic amino acids within transmembrane segment bundles. Herein, we examined the intrinsic transport function of WzxPafollowing its purification and reconstitution in phospholipid liposomes. WzxPawas capable of mediating anion flux, consistent with its cationic interior. This flux was electrogenic and modified by extraliposomal pH. Mutation of the above-mentioned acidic residues (E61, D269, and D359) reduced proton (H+)-modified anion flux, showing the role of these amino acid side chains in H+-dependent transport. Wzx also mediated acidification of the proteoliposome interior in the presence of an outward anion gradient. These results indicate H+-dependent gating and H+uptake by WzxPaand allow for the first H+-dependent antiport mechanism to be proposed for lipid-linked oligosaccharide translocation across the bacterial IM.IMPORTANCEMany bacterial cell surface polysaccharides that are important for survival and virulence are synthesized at the periplasmic leaflet of the inner membrane (IM) using precursors produced in the cytoplasm. Wzx flippases are responsible for translocation of lipid-linked sugar repeat units across the IM and had been previously suggested to simply facilitate passive substrate diffusion. Through our characterization of purified Wzx in a reconstitution system described herein, we have observed protein-dependent intrinsic transport producing a change in the electrical potential of the system, with H+identified as the coupling ion. These results provide the first evidence for coupled (i.e., secondary active) transport by these proteins and, in conjunction with structural data, allow for an antiport mechanism to be proposed for the directed transport of lipid-linked sugar substrates across the IM. These findings bring our understanding of lipid-linked polysaccharide transporter proteins more in line with the efflux pumps to which they are evolutionarily related.