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Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which regulates chloride and water transport across all epithelia and affects multiple organs, including the lungs. Here we report an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction-coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof of concept, treatment of CF patient induced pluripotent stem cell-derived epithelial cells with a small-molecule compound to correct for the common CF processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein. Our study provides a method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing. [ABSTRACT FROM AUTHOR]

Objective Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using 31P-magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high-intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end-exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle. Methods and Results We studied CFTR expression in human skeletal muscle by Western blot with anti-CFTR antibody (Ab) L12B4 and demonstrated a single band with expected molecular weight of 168kDa. We isolated the cDNA by reverse transcription polymerase chain reaction and directly sequenced a 975bp segment (c. 3,600-4,575) that was identical to the human CFTR sequence. We showed punctate staining of CFTR in sarcoplasm and sarcolemma by immunofluorescence microscopy with L12B4 Ab and secondary Alexa 488-labeled Ab. We confirmed CFTR expression in the sarcotubular network and sarcolemma by electron microscopy, using immunogold-labeled anti-CFTR Ab. We observed activation of CFTR Cl− channels with iodide efflux, on addition of forskolin, 3-isobutyl-1-methyl-xanthine, and 8-chlorphenylthio-cyclic adenosine monophosphate, in wild-type C57BL/6J isolated muscle fibers in contrast to no efflux from mutant F508del-CFTR muscle. Interpretation We speculate that a defect in sarcoplasmic reticulum CFTR Cl− channels could alter the electrochemical gradient, causing dysregulation of Ca2+ homeostasis, for example, ryanodine receptor or sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatases essential to excitation-contraction coupling leading to exercise intolerance and muscle weakness in CF. ANN NEUROL 2010 [ABSTRACT FROM AUTHOR]

The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. Clearly, an understanding of the structure–function relationships of these proteins will be critical in defining the molecular mechanisms underlying disease pathogenesis. The X-ray crystal structure of prokaryotic ClC proteins provides an exquisite template with which to model molecular aspects of eukaryotic ClC protein function. The dimeric structure of these proteins highlights the pivotal importance of intermolecular interactions in the modulation of channel/transporter activity, while mutagenesis studies implicate a crucial role for intrinsic interdomain interactions in regulated function. In this review, we will initially focus on the channel forming members of this family and discuss interactions within homodimeric channel complexes important for gating. Finally, with regard to both channel and transporter family members, we will discuss the multiple heteromeric interactions which occur with cytosolic proteins, and the putative functional impact of these interactions. [ABSTRACT FROM AUTHOR]

Background: Cystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. Methods: Small molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. Results: We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. Conclusion: Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD. [ABSTRACT FROM AUTHOR]

• Recent in vitro data suggested that ELX/TEZ/IVA increases N1303K-CFTR function. • 8 pwCF that started off label ELX/TEZ/IVA and had significant clinical response, with increase in FEV1 and BMI, though sweat chloride remained in the pathologic value. • Although not listed as ELX/TEZ/IVA responsive mutatoin, PwCF carrying the N1303K mutation may have significant clinical benefit by this treatment. Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) significantly improves health outcomes in people with cystic fibrosis (pwCF) carrying one or two F508del mutations. According to in vitro assays performed in FRT cells, 178 additional mutations respond to ELX/TEZ/IVA. The N1303K mutation is not included in this list of mutations. Recent in vitro data suggested that ELX/TEZ/IVA increases N1303K-CFTR activity. Based on the in vitro response, eight patients commenced treatment with ELX/TEZ/IVA. Two homozygotes; and six compound heterozygotes N1303K/nonsense or frameshift mutation pwCF were treated off label with ELX/TEZ/IVA. Clinical data before and 8 weeks after starting treatment were prospectively collected. The response to ELX/TEZ/IVA was assessed in intestinal organoids derived from 5 study patients and an additional patient carrying N1303K that is not receiving treatment. Compared to the values before commencing treatment, mean forced expiratory volume in 1 second increased by 18.4 percentage points and 26.5% relative to baseline, mean BMI increased by 0.79 Kg/m2, and mean lung clearance index decreased by 3.6 points and 22.2%. There was no significant change in sweat chloride. Nasal potential difference normalized in four patients and remained abnormal in three. Results in 3D intestinal organoids and 2D nasal epithelial cultures showed a response in CFTR channel activity. This report supports the previously reported in vitro data, performed in human nasal and bronchial epithelial cells and intestinal organoids, that pwCF who carry the N1303K mutation have a significant clinical benefit by ELX/TEZ/IVA treatment. [ABSTRACT FROM AUTHOR]

• Predictive tools to identify best responders to modulators appear cost-effective. • Predictive tools may save $931 K to $1.1 M per quality adjusted life year lost. • Predictive tools could optimize health benefits and reduce high healthcare costs. Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator therapies show variable efficacy for patients with CF. Patient-derived predictive tools may identify individuals likely to respond to CFTRs, but are not in routine use. We aimed to determine the cost-utility of predictive tool-guided treatment with CFTRs as add-on to standard of care (SoC) for individuals with CF. This economic evaluation compared two strategies using an individual level simulation: (i) Treat All , where all patients received CFTRs plus SoC and (ii) Test→Treat , where patients who tested positive on predictive tools received CFTRs plus SoC and those who tested negative received SoC only. We simulated 50,000 individuals over their lifetime, and estimated costs (2020 CAD) per quality-adjusted life year (QALY) from the healthcare payer's perspective, discounted at 1.5% annually. The model was populated using Canadian CF registry data and published literature. Probabilistic and deterministic sensitivity were conducted. The Treat All and Test→Treat and strategies yielded 22.41 and 21.36 QALYs, and cost $4.21 M and $3.15 M respectively. Results of probabilistic sensitivity analysis showed that Test→Treat was highly cost-effective compared to Treat All in 100% of simulations at cost-effectiveness thresholds as high as $500,000 per QALY. Test→Treat may save between $931 K to $1.1 M per QALY lost, depending on sensitivity and specificity of predictive tools. The use of predictive tools could optimize the health benefits of CFTR modulators while reducing costs. Our findings support the use of pre-treatment predictive testing and may help inform coverage and reimbursement policies for individuals with CF. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling. [ABSTRACT FROM AUTHOR]

Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3–6 weeks in order to understand genetic determinants of disease and precision medicine. [ABSTRACT FROM AUTHOR]

It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del (n = 31) or W1282X (n = 3), or heterozygous for Class III mutations G551D or G178R (n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X. [ABSTRACT FROM AUTHOR]

Membrane permeable bi-arsenical dyes, such as fluorescein arsenical hairpin (FlAsH), bind tetracysteine-tagged-proteins emitting fluorescence in-vivo. This fluorescence method (developed by R. Tsien) provides a tool to evaluate trafficking pathways in living cells. We generated a tetracysteine-tagged Cystic Fibrosis Transmembrane Regulator (CFTR) protein and studied its expression with the long-term goal of using this protein to study its trafficking during endocytosis. The tetracysteine-tagged CFTR (CFTR-TC) was predominantly expressed in both BHK and in Cos-7 cells as the immature core-glycosylated form (band B) rather than the mature glycosylated band C. Iodide efflux assays of CFTR-TC's function as a chloride channel at the cell surface revealed an insignificant response after cAMP stimulation, likely reflecting its low band C expression. Interestingly, 1 hour FlAsH pretreatment evoked a mean response difference of -16.0 mV versus untreated (one-way ANOVA p<0.05), suggesting increased band C expression. Our results suggest that FlAsH rescues the surface expression of CFTR-TC and studies are ongoing to visualize its localization and the regulation of its trafficking by proteins involved in the endocytic pathway. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl concentration ([Cl]). Patch clamp current recordings show that a rise in [Cl] stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl] sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl] decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl] is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues. [ABSTRACT FROM AUTHOR]

Although bile duct disorders are well-recognized causes of liver disease, the molecular and cellular events leading to biliary dysfunction are poorly understood. To enable modeling and drug discovery for biliary disease, we describe a protocol that achieves efficient differentiation of biliary epithelial cells (cholangiocytes) from human pluripotent stem cells (hPSCs) through delivery of developmentally relevant cues, including NOTCH signaling. Using three-dimensional culture, the protocol yields cystic and/or ductal structures that express mature biliary markers, including apical sodium-dependent bile acid transporter, secretin receptor, cilia and cystic fibrosis transmembrane conductance regulator (CFTR). We demonstrate that hPSC-derived cholangiocytes possess epithelial functions, including rhodamine efflux and CFTR-mediated fluid secretion. Furthermore, we show that functionally impaired hPSC-derived cholangiocytes from cystic fibrosis patients are rescued by CFTR correctors. These findings demonstrate that mature cholangiocytes can be differentiated from hPSCs and used for studies of biliary development and disease. [ABSTRACT FROM AUTHOR]

• Efficient antisense oligonucleotides (ASOs) promote skipping of exon 23. • The lead ASOs promote generation of mature CFTR protein lacking exon 23. • The lead ASOs together with CFTR modulators restore CFTR function. • The lead ASOs are specific for CFTR alleles harboring the W1282X mutation. • The W1282X antisense-based splicing modulation is progressing to drug development. Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23. ∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient. ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient. The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23–2 to proof-of-concept clinical study. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause loss of function of the CFTR channel on the apical surface of epithelial cells. The major CF-causing mutation, F508del-CFTR, is misfolded, retained in the endoplasmic reticulum, and degraded. Small molecule corrector compounds have been identified using high throughput screens, which partially rescue the trafficking defect of F508del-CFTR, allowing a fraction of the mutant protein to escape endoplasmic reticulum retention and traffic to the plasma membrane, where it exhibits partial function as a cAMP-regulated chloride channel. A subset of such corrector compounds binds directly to the mutant protein, prompting the hypothesis that they rescue the biosynthetic defect by inducing improved protein conformation. We tested this hypothesis directly by evaluating the consequences of a corrector compound on the conformation of each nucleotide binding domain (NBD) in the context of the full-length mutant protein in limited proteolytic digest studies. Interestingly, we found that VRT-325 was capable of partially restoring compactness in NBD1. However, VRT-325 had no detectable effect on the conformation of the second half of the molecule. In comparison, ablation of the di-arginine sequence, R553XR555 (F508del-KXK-CFTR), modified protease susceptibility of NBD1, NBD2, and the full-length protein. Singly, each intervention led to a partial correction of the processing defect. Together, these interventions restored processing of F508del-CFTR to near wild type. Importantly, however, a defect in NBD1 conformation persisted, as did a defect in channel activation after the combined interventions. Importantly, this defect in channel activation can be fully corrected by the addition of the potentiator, VX-770. [ABSTRACT FROM AUTHOR]

The involvement of several members of the chloride channel (ClC) family of membrane proteins in human disease highlights the need to define the mechanisms underlying their function and the consequences of disease-causing mutations. Despite the utility of high-resolution structural models, our understanding of the molecular basis for function of the chloride channels and transporters in the family remains incomplete. In this review, we focus on recent discoveries regarding molecular mechanisms underlying the regulated chloride:proton antiporter activity of ClC-5, the protein mutated in the Dent’s disease—a kidney disease presenting with proteinuria and renal failure in severe cases. We discuss the putative role of ClC-5 in receptor-mediated endocytosis and protein uptake by the proximal renal tubule and the possible molecular and cellular consequences of disease-causing mutations. However, validation of these models will require future study of the intrinsic function of this transporter, in situ, in the membranes of recycling endosomes in proximal tubule epithelial cells. [ABSTRACT FROM AUTHOR]

• The Rio-CF study evaluated the efficacy and safety of riociguat in adults with CF. • Rio-CF was halted due to lack of efficacy and a changing therapeutic CF landscape. • No safety concerns were raised during Rio-CF. • There was no evidence that riociguat could be valid treatment option for CF. Riociguat is a first-in-class soluble guanylate cyclase stimulator for which preclinical data suggested improvements in cystic fibrosis transmembrane conductance regulator (CFTR) function. This international, multicenter, two-part, Phase II study of riociguat enrolled adults with cystic fibrosis (CF) homozygous for Phe508del CFTR. Part 1 was a 28-day, randomized, double-blind, placebo-controlled study in participants not receiving CFTR modulator therapy. Twenty-one participants were randomized 1:2 to placebo or oral riociguat (0.5 mg three times daily [tid] for 14 days, increased to 1.0 mg tid for the subsequent 14 days). The primary and secondary efficacy endpoints were change in sweat chloride concentration and percent predicted forced expiratory volume in 1 second (ppFEV 1), respectively, from baseline to Day 14 and Day 28 with riociguat compared with placebo. Riociguat did not alter CFTR activity (change in sweat chloride) or lung function (change in ppFEV 1) at doses up to 1.0 mg tid after 28 days. The most common drug-related adverse event (AE) was headache occurring in three participants (21%); serious AEs occurred in one participant receiving riociguat (7%) and one participant receiving placebo (14%). This safety profile was consistent with the underlying disease and the known safety of riociguat for its approved indications. The Rio-CF study was terminated due to lack of efficacy and the changing landscape of CF therapeutic development. The current study⁠, within its limits of a small sample size, did not provide evidence that riociguat could be a valid treatment option for CF. Clinical trial registration number: NCT02170025. [ABSTRACT FROM AUTHOR]

The rare Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, c.1826A > G (H609R) and c.3067_3072delATAGTG (I1023_V1024del), are associated with severe lung disease. Despite the existence of four CFTR targeted therapies, none have been approved for individuals with these mutations because the associated molecular defects were not known. In this study we examined the consequences of these mutations on protein processing and channel function in HEK293 cells. We found that, similar to F508del, H609R and I1023_V1024del-CFTR exhibited reduced protein processing and altered channel function. Because the I1023_V1024del mutation can be linked with the mutation, I148T, we also examined the protein conferred by transfection of a plasmid bearing both mutations. Interestingly, together with I148T, there was no further reduction in channel function exhibited by I1023-V1024del. Both H609R and I1023_V1024del failed to exhibit significant correction of their functional expression with lumacaftor and ivacaftor. In contrast, the triple modulator combination found in TRIKAFTATM, i.e., tezacaftor, elexacaftor and ivacaftor rescued trafficking and function of both of these mutants. These in-vitro findings suggest that patients harbouring H609R or I1023_V1024del, alone or with I148T, may benefit clinically from treatment with TRIKAFTATM. [ABSTRACT FROM AUTHOR]

Cholangiocytes form an intricate network of bile ducts to enable proper liver function; yet, recapitulating human stem cell differentiation to cholangiocytes in vitro requires Notch signaling and soluble ligands do not activate the Notch pathway. To overcome these limitations, jagged1 is immobilized on a chemically defined hyaluronan to specifically differentiate human embryonic stem cell‐derived hepatoblasts to cholangiocytes. Hepatoblasts cultured on the jagged1‐hydrogels upregulate Notch target genes and express key cholangiocyte markers including cystic fibrosis transmembrane conductance regulator. Moreover, cholangiocytes adopt morphological changes that resemble liver biliary structures. To emulate natural biliary system development, a new strategy is developed to achieve spatiotemporal control over the Jagged1–Notch2 interaction: jagged1 is first caged with a photocleavable streptavidin and then it is uncaged photochemically to restore the biological function of Jagged1, which is confirmed with Notch2 activation in a fluorescent reporter cell line. Moreover, the differentiation of human embryonic stem cell‐derived hepatoblasts to cholangiocytes is temporally controlled with photochemical uncaging of this streptavidin‐Jagged1‐immobilized hyaluronan hydrogel. This strategy defines a framework to control protein signaling in time and space and specifically for Notch signaling for ultimate use in regenerative medicine strategies of the liver. [ABSTRACT FROM AUTHOR]

• Rare Class 2 CFTR mutations rescued by strategic modulator combinations. • Modulator responses vary across cell types. • A dual acting (corrector-potentiator) compound contributes to functional rescue of N1303K. F508del is prototypical of Class 2 CFTR mutations associated with protein misprocessing and reduced function. Corrector compounds like lumacaftor partially rescue the processing defect of F508del-CFTR whereas potentiators like ivacaftor, enhance its channel activity once trafficked to the cell surface. We asked if emerging modulators developed for F508del-CFTR can rescue Class 2 mutations previously shown to be poorly responsive to lumacaftor and ivacaftor. Rescue of mutant CFTRs by the correctors: AC1, AC2–1 or AC2–2 and the potentiator, AP2, was studied in HEK-293 cells and in primary human nasal epithelial (HNE) cultures, using a membrane potential assay and Ussing chamber, respectively. In HEK-293 cells, we found that a particular combination of corrector molecules (AC1 plus AC2–1) and a potentiator (AP2) was effective in rescuing both the misprocessing and reduced function of M1101K and G85E respectively. These findings were recapitulated in patient-derived nasal cultures, although another corrector combination, AC1 plus AC2–2 also improved misprocessing in these primary tissues. Interestingly, while this corrector combination only led to a modest increase in the abundance of mature N1303K-CFTR it did enable its functional expression in the presence of the potentiator, AP2, in part, because the nominal corrector, AC2–2 also exhibits potentiator activity. Strategic combinations of novel modulators can potentially rescue Class 2 mutants thought to be relatively unresponsive to lumacaftor and ivacaftor. [ABSTRACT FROM AUTHOR]

Chronic infection and inflammation are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. ORKAMBI® (Lumacaftor-Ivacaftor) is an approved combination therapy for Cystic Fibrosis (CF) patients bearing the most common mutation, F508del, in the cystic fibrosis conductance regulator (CFTR) protein. It has been previously shown that ORKAMBI®-mediated rescue of CFTR is reduced by a pre-existing Pseudomonas aeruginosa infection. Here, we show that the infection of F508del-CFTR human bronchial epithelial (HBE) cells with lab strain and four different clinical strains of P. aeruginosa, isolated from the lung sputum of CF patients, decreases CFTR function in a strain-specific manner by 48 to 88%. The treatment of infected cells with antibiotic tobramycin or cationic antimicrobial peptide 6K-F17 was found to decrease clinical strain bacterial growth on HBE cells and restore ORKAMBI®-mediated rescue of F508del-CFTR function. Further, 6K-F17 was found to downregulate the expression of pro-inflammatory cytokines, interleukin (IL)-8, IL-6, and tumor necrosis factor-α in infected HBE cells. The results provide strong evidence for a combination therapy approach involving CFTR modulators and anti-infectives (i.e., tobramycin and/or 6K-F17) to improve their overall efficacy in CF patients. [ABSTRACT FROM AUTHOR]

• Recombinant W1282X-CFTR protein rescued with corrector combination and potentiator. • In nasal cultures, W1282X-CFTR rescue requires inhibition of nonsense mediated decay. • Response to this quadruple combination is variable across cultures from 4 people. The nonsense mutation, c.3846G>A (aka: W1282X-CFTR) leads to a truncated transcript that is susceptible to nonsense-mediated decay (NMD) and produces a shorter protein that is unstable and lacks normal channel activity in patient-derived tissues. However, if overexpressed in a heterologous expression system, the truncated mutant protein has been shown to mediate CFTR channel function following the addition of potentiators. In this study, we asked if a quadruple combination of small molecules that together inhibit nonsense mediated decay, stabilize both halves of the mutant protein and potentiate CFTR channel activity could rescue the functional expression of W1282X-CFTR in patient derived nasal cultures. We identified the CFTR domains stabilized by corrector compounds supplied from AbbVie using a fragment based, biochemical approach. Rescue of the channel function of W1282X.-CFTR protein by NMD inhibition and small molecule protein modulators was studied using a bronchial cell line engineered to express W1282X and in primary nasal epithelial cultures derived from four patients homozygous for this mutation. We confirmed previous studies showing that inhibition of NMD using the inhibitor: SMG1i, led to an increased abundance of the shorter transcript in a bronchial cell line. Interestingly, on top of SMG1i, treatment with a combination of two new correctors developed by Galapagos/AbbVie (AC1 and AC2-2, separately targeting either the first or second half of CFTR and promoting assembly, significantly increased the potentiated channel activity by the mutant in the bronchial epithelial cell line and in patient-derived nasal epithelial cultures. The average rescue effect in primary cultures was approximately 50% of the regulated chloride conductance measured in non-CF cultures. These studies provide the first in-vitro evidence in patient derived airway cultures that the functional defects incurred by W1282X, has the potential to be effectively repaired pharmacologically. [ABSTRACT FROM AUTHOR]

Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell type–specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh172 to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10 μM induced HEE organoid swelling by 20% at 16 h, while 5 and 10 μM CFTRinh172 treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70-kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. These results facilitate progress in elucidating how CFTR-dependent cellular processes impair fertility in CF. [ABSTRACT FROM AUTHOR]

F508del‐cystic fibrosis transmembrane conductance regulator (CFTR) is the major mutant responsible for cystic fibrosis (CF). ORKAMBI®, approved for patients bearing this mutant, contains lumacaftor (VX‐809) that partially corrects F508del‐CFTR's processing defect and ivacaftor (VX‐770) that potentiates its defective channel activity. Unfortunately, the clinical efficacy of ORKAMBI® is modest, highlighting the need to understand how the small molecules work so that superior compounds can be developed. Because, human CFTR (hCFTR) and zebrafish Cftr (zCftr) are structurally conserved as determined in recent cryo‐EM structural models, we hypothesized that the consequences of the major mutation and small molecule modulators would be similar for the two species of protein. As expected, like the F508del mutation in hCFTR, the homologous mutation in zCftr (F507del) is misprocessed, yet not as severely as the human mutant and this defect was restored by low‐temperature (27°C) culture conditions. After rescue to the cell surface, F507del‐zCftr exhibited regulated channel activity that was potentiated by ivacaftor. Surprisingly, lumacaftor failed to rescue misprocessing of the F507del‐zCftr at either 37 or 27°C suggesting that future comparative studies with F508del‐hCFTR would provide insight into its structure: function relationships. Interestingly, the robust rescue of F508del‐zCftr at 27°C and availability of methods for in vivo screening in zebrafish present the opportunity to define the cellular pathways underlying rescue. [ABSTRACT FROM AUTHOR]

Abstract Cystic Fibrosis (CF) is a genetic disorder caused by loss-of-function mutations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Ivacaftor ( 1 ) was the first therapeutic approved for the treatment of CF that is able to restore gating activity to certain CFTR variants although the mechanism of action is poorly understood. Herein we describe the synthesis of a photoaffinity labelling (PAL) probe ( 2 ) based on the structure of ivacaftor incorporating a photoreactive diazirine moiety for use in labelling studies designed to identify the binding site for ivacaftor on mutant CFTR. The PAL probe 2 retained potentiation activity, with a potency similar to 1 , using a Fluorescent Imaging Plate Reader (FLIPR®) assay measuring ion conductance potentiation of wild type (Wt)-CFTR. Photolabelling experiments with human serum albumin (HSA) as a model protein have shown that probe 2 can label HSA in a manner consistent with observed and predicted binding. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air–liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases. [ABSTRACT FROM AUTHOR]

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations – glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis. [ABSTRACT FROM AUTHOR]

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulinmediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation- regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinaseA(PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation. [ABSTRACT FROM AUTHOR]

Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) gene. The most common mutation, deletion of phenylalanine 508 (F508del), disrupts tertiary assembly, causing protein misprocessing and loss of CFTR function in epithelial tissues. Lumacaftor (VX-809) is a Class 1 corrector molecule shown to partially rescue misprocessing of F508del and together with the potentiator of channel activity: ivacaftor (VX-770) has been approved for treatment of CF patients homozygous for the F508del mutation. The specificity of these modulators for CFTR is thought to be conferred through direct binding. Trimethylangelicin (TMA) is a distinct small molecule modulator, previously shown to exhibit both corrector and potentiator activities. We were prompted to determine if TMA also mediates these activities by direct binding. Interestingly, we found that like VX-770, TMA was effective in enhancing anion efflux mediated by purified WT-CFTR reconstituted in phospholipid liposomes. Furthermore, like VX-809, TMA was effective in stabilizing the functional expression of CFTR lacking the regulatory “R” domain or second nucleotide-binding domain (NBD2). The smallest domain that was stabilized by TMA binding was the first membrane-spanning domain (MSD1) as previously observed for VX-809. Together, our findings support the claim that TMA binds directly to CFTR, and despite its distinct chemical structure, shares similar mechanisms as VX-770 and VX-809 to potentiate and stabilize CFTR, respectively. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking. [ABSTRACT FROM AUTHOR]

Summary: The most common mutation causing cystic fibrosis (CF), F508del, impairs conformational maturation of CF transmembrane conductance regulator (CFTR), thereby reducing its functional expression on the surface of epithelia. Corrector compounds including C18 (VRT-534) and VX-809 have been shown to partially rescue misfolding of F508del-CFTR and to enhance its maturation and forward trafficking to the cell surface. Now, we show that there is an additional action conferred by these compounds beyond their role in improving the biosynthetic assembly. In vitro studies show that these compounds bind directly to the metastable, full-length F508del-CFTR channel. Cell culture and patient tissue-based assays confirm that in addition to their cotranslational effect on folding, certain corrector compounds bind to the full-length F508del-CFTR after its partial rescue to the cell surface to enhance its function. These findings may inform the development of alternative compounds with improved therapeutic efficacy. [Copyright &y& Elsevier]

Mutations in the CLCN5 (chloride channel, voltage-sensitive 5) gene cause Dent's disease because they reduce the functional expression of the ClC-5 chloride/proton transporter in the recycling endosomes of proximal tubule epithelial cells. The majority (60%) of these disease-causing mutations in ClC-5 are misprocessed and retained in the ER (endoplasmic reticulum). Importantly, the structural basis for misprocessing and the cellular destiny of such ClC-5 mutants have yet to be defined. A ClC-5 monomer comprises a short N-terminal region, an extensive membrane domain and a large C-terminal domain. The recent crystal structure of a eukaryotic ClC (chloride channel) transporter revealed the intimate interaction between the membrane domain and the C-terminal region. Therefore we hypothesized that intramolecular interactions may be perturbed in certain mutants. In the present study we examined two misprocessed mutants: C221R located in the membrane domain and R718X,which truncates the C-terminal domain. Both mutants exhibited enhanced protease susceptibility relative to the normal protein in limited proteolysis studies, providing direct evidence that they are misfolded. Interestingly, the membrane-localized mutation C221R led to enhanced protease susceptibility of the cytosolic N-terminal region, and the C-terminal truncation mutation R718X led to enhanced protease susceptibility of both the cytosolic C-terminal and the membrane domain. Together, these studies support the idea that certain misprocessing mutations alter intramolecular interactions within the full-length ClC-5 protein. Further, we found that these misfolded mutants are polyubiquitinated and targeted for proteasomal degradation in the OK (opossum kidney) renal epithelial cells, thereby ensuring that they do not elicit the unfolded protein response. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a non conventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 Å crystal structure of AlgE, which reveals a monomeric 18-stranded β-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 (ΔT8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or ΔL2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with ΔT8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems. [ABSTRACT FROM AUTHOR]

ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation. [ABSTRACT FROM AUTHOR]

Abstract: The intracellular delivery of a diverse array of cargos can be mediated by conjugation to cell-penetrating peptides (CPPs). To date, delivery of cargos into the cytosol via CPPs has been measured indirectly and normally, has been inferred from changes in biological activity. We describe a novel method to directly assay CPP-mediated delivery of peptide cargo into the cytosol, and use this method to define the kinetics of this process. The CPP and the cargo are differentially labeled with the fluorophores FAM (carboxyfluorescein), and TAMRA (carboxytetramethylrhodamine) respectively, and coupled via a disulfide bond to promote quenching of FAM fluorescence by the proximal TAMRA. Delivery of the peptide pair to cells produces an increase in FAM fluorescence within 10 min, consistent with its rapid transfer into the reducing environment of the cytosol, separation of the two components, and concomitant dequenching. The fluorescence-based assay described here can thus be used to select a CPP module that is optimized for efficient delivery of particular cargos designed to modify molecular targets in the cytosol. [Copyright &y& Elsevier]

Summary: The cystic fibrosis (CF)-causing mutant, deltaF508-CFTR, is misfolded and fails to traffic out of the endoplasmic reticulum (ER) to the cell surface. Introduction of second site mutations that disrupt a diarginine (RXR)-based ER retention motif in the first nucleotide binding domain rescues the trafficking defect of deltaF508-CFTR, supporting a role for these motifs in mediating ER retention of the major mutant. To determine if these RXR motifs mediate retention of the native deltaF508-CFTR protein in situ, we generated peptides that mimic these motifs and should antagonize mistrafficking mediated via their aberrant exposure. Here we show robust rescue of deltaF508-CFTR in cell lines and in respiratory epithelial tissues by transduction of RXR motif-mimetics, showing that abnormal accessibility of this motif is a key determinant of mistrafficking of the major CF-causing mutant. [Copyright &y& Elsevier]

Abstract: CFTR is a member of the ABC (ATP binding cassette) superfamily of transporters. It is a multidomain membrane protein, which utilizes ATP to regulate the flux of its substrate through the membrane. CFTR is distinct in that it functions as a channel and it possesses a unique regulatory R domain. There has been significant progress in understanding the molecular basis for CFTR activity as an ATPase. The dimeric complex of NBD structures seen in prokaryotic ABC transporters, together with the structure of an isolated CF-NBD1, provide a unifying molecular template to model the structural basis for the ATPase activity of CFTR. The dynamic nature of the interaction between the NBDs and the R domain has been revealed in NMR studies. On the other hand, understanding the mechanisms mediating the transmission of information from the cytosolic domains to the membrane and the channel gate of CFTR remains a central challenge. [Copyright &y& Elsevier]

Recent crystal structures of the CorA Mg2+ transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understand- ing the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg2tselective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic res- idues found in the short periplasmic loop are not essential for TmCorA function or Mg2~ selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alrl-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted. [ABSTRACT FROM AUTHOR]

The chloride channel, ClC-2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC-2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC-2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC-2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC-2 internalization by endocytosis. Cell surface expression of the disease-causing mutant: G715E, thought to lack wild-type nucleotide binding affinity, is similarly affected, suggesting that ATP-depletion modifies the function of proteins in the endocytic pathway rather than ClC-2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC-2 is internalized via dynamin-dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant-negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5-associated vesicles and recycling of ClC-2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC-2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel. J. Cell. Physiol. 214:273–280, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins. Recently, crystal structures of intact, prokaryotic members of this family have been described. These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs). In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis. In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together. We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity. Co-assembly of NBD1 and NBD2 exhibited a 2–3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (Vmax). These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1H/2H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site. [ABSTRACT FROM AUTHOR]

Mutations in the gene coding for the chloride channel ClC-5 cause Dent's disease, a disease associated with proteinuria and renal stones. Studies in ClC-5 knockout mice suggest that this phenotype is related to defective endocytosis of low molecular weight proteins and membrane proteins by the renal proximal tubule. In this study, confocal micrographs of proximal tubules and cultured epithelial cells revealed that the related protein ClC-4 is expressed in endosomal membranes suggesting that this channel may also contribute to the function of this organelle. In support of this hypothesis, specific disruption of endogenous ClC-4 expression by transfection of ClC-4 antisense cDNA acidified endosomal pH and altered transferrin trafficking in cultured epithelial cells to the same extent as the specific disruption of ClC-5. Both channels can be co-immunoprecipitated, arguing that they may partially contribute to endosomal function as a channel complex. These studies prompt future investigation of the role of ClC-4 in renal function in health and in Dent's disease. Future studies will assess whether the severity of Dent's disease relates not only to the impact of particular mutations on ClC-5 but also on the consequences of those mutations on the functional expression of CIC-4. [ABSTRACT FROM AUTHOR]

Shows that expression of ClC-2 chloride channel at the cell surface may be regulated via an interaction with the dynein motor complex. Co-immunoprecipitation of the dynein intermediate chain with ClC-2 from hippocampal membranes; Perturbation of ClC-2 localization with the disruption of dynein motor function.

Examines the function of cystic fibrosis transmembrane conductase regulator (CFTR). Use of adenosine triphosphatase activity to regulate opening and closing of channel gate; Need for quaternary structure to mediate conduction and catalysis; Size of CFTR dimers.

Demonstrates expression of cystic fibrosis transmembrane conductance regulator (CFTR) message and protein in neuroepithelial body (NEB) cells of rabbit neonatal lungs. CFTR localization in lung cells as determined by confocal microscopy; Expression of mRNA for tryptophan hydroxylase and CFTR in NEB using RT-PCR.

Presents a study which demonstrated expression of cystic fibrosis transmembrane conductance regulator message and protein in neuroepithelial body cells of rabbit neonatal lungs. Methods; Results and discussion.