CFTR Cl Research Articles

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function.

The combination of elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) reverses the primary defect in Cystic Fibrosis (CF) by improving CFTR mediated Cl- and HCO3- secretion by airway epithelial cells (AEC), leading to improved lung function and less frequent exacerbations and hospitalizations. However, studies have shown that CFTR modulators like ivacaftor, a component of ETI, has numerous effects on CF cells beyond improved CFTR channel function. Because little is known about the effect of ETI on CF AEC gene expression we exposed primary human AEC to ETI for 48 hours and interrogated the transcriptome by RNA-seq and qPCR. ETI increased CFTR Cl- secretion, and defensin gene expression (DEFB1) an observation consistent with reports of decreased bacterial burden in the lungs of people with CF (pwCF). ETI decreased MMP10 and MMP12 gene expression, suggesting that ETI may reduce proteolytic induced lung destruction in CF. ETI also reduced the expression of the stress response gene heme oxygenase (HMOX1). qPCR analysis confirmed DEFB1, HMOX1, MMP10 and MMP12 gene expression results observed by RNA-seq. Gene pathway analysis revealed that ETI decreased inflammatory signaling, cellular proliferation and MHC Class II antigen presentation. Collectively, these findings suggest that the clinical observation that ETI reduces lung infections in pwCF is related in part to drug induced increases in DEFB1, and that ETI may reduce lung damage by reducing MMP10 and MMP12 gene expression. Moreover, pathway analysis also identified several other genes responsible for the ETI induced reduction in inflammation observed in pwCF.

The Secondary Bile acid, Lithocholic Acid, Inhibits Cl− Secretion Across Colonic Epithelial Cells in Vitro

Lithocholic acid (LCA) is a secondary bile acid formed in the colon by bacterial metabolism of chenodeoxycholic acid. Our previous studies have shown the bile acid to be protective under conditions of colonic inflammation. Here, we sought to determine if LCA might also play a role in regulating fluid and electrolyte transport. T84 cell monolayers were mounted in Ussing chambers for measurements of Cl− secretion, the primary driving force for fluid secretion into the colon. qRT-PCR and Western blotting were used to analyze mRNA and protein expression. Results were expressed as mean ± SEM and data were analyzed by one way ANOVA and Tukey’s post hoc test or by mixed-effects analysis and Dunnett's post hoc test. To assess the effects of LCA on CFTR promoter activity, we used a luciferase promoter/reporter system, where HEK293 cells were transfected with a plasmid containing the firefly luciferase gene under control of the CFTR promoter. Co-transfections were carried out with 2 additional plasmids expressing either the nuclear bile acid receptor, FXR, or its dimerization partner, RXR. Pretreatment of T84 cell monolayers with LCA inhibited subsequent Cl− secretory responses to the cAMP-dependent agonist, forskolin (FSK; 10 μM), in a concentration (1 – 10 μM) and time-dependent (3 - 24 hrs) manner. Maximal effects of LCA were observed at a concentration of 10 μM after treatment for 24 hrs, when responses to FSK were reduced to 50.9 ± 8.5% of those in controls (n = 6; p < 0.01). LCA (10 μM; 24 hrs) also inhibited responses to the Ca2+-dependent secretagogues, thapsigargin (2 μM) and histamine (100 μM), by 59.4 ± 2.4% (n = 4; p < 0.001) and 52.2 ± 1.9% (n = 5; p < 0.001), respectively. In further experiments, using nystatin-permeabilized T84 monolayers to isolate apical Cl− conductances, LCA (10 μM; 24 hrs) reduced FSK-stimulated responses to 72.7 ± 6.6% (n = 17; p < 0.001) of those in control cells. Analysis of CFTR expression, the primary exit pathway for Cl− in colonic epithelial cells, revealed that LCA treatment reduced mRNA and protein expression of the channel to 0.65 ± 0.05 fold (n = 7; p < 0.01) and 0.43 ± 0.06 fold (n = 6; p < 0.001), respectively. In CFTR promoter assays, LCA (10 μM) reduced CFTR promoter activity to 0.7 ± 0.02 fold of that in control cells (n = 5; p < 0.01), with co-expression of FXR being required for this effect to be observed. Finally, while LCA activated both FXR and the vitamin D receptor (VDR) in T84 cells, its effects in downregulating CFTR expression and Cl− conductances were mimicked by the FXR agonist, GW4064, but not by the VDR agonist, calcitriol. In conclusion, LCA, at physiologically relevant concentrations, inhibits Cl− secretion across colonic epithelial cells, likely through a mechanism involving FXR activation and inhibition of CFTR expression. These data add to the growing pool of knowledge regarding important regulatory actions of LCA in the colon and highlight its potential role as a target for the treatment of intestinal disorders. This work was supported by a grant from Science Foundation Ireland. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Molecular mechanisms of dexamethasone actions in COVID-19: Ion channels and airway surface liquid dynamics

The COVID-19 pandemic has been a global health crisis of unprecedented magnitude. In the battle against the SARS-CoV-2 coronavirus, dexamethasone, a widely used corticosteroid with potent anti-inflammatory properties, has emerged as a promising therapy in the fight against severe COVID-19.Dexamethasone is a synthetic glucocorticoid that exerts its therapeutic effects by suppressing the immune system and reducing inflammation. In the context of COVID-19, the severe form of the disease is often characterized by a hyperactive immune response, known as a cytokine storm. Dexamethasone anti-inflammatory properties make it a potent tool in modulating this exaggerated immune response.Lung inflammation may lead to excessive fluid accumulation in the airways which can reduce gas exchange and mucociliary clearance. Pulmonary oedema and flooding of the airways are hallmarks of severe COVID-19 lung disease. The volume of airway surface liquid is determined by a delicate balance of salt and water secretion and absorption across the airway epithelium. In addition to its anti-inflammatory actions, dexamethasone modulates the activity of ion channels which regulate electrolyte and water transport across the airway epithelium. The observations of dexamethasone activation of sodium ion absorption via ENaC Na+ channels and inhibition of chloride ion secretion via CFTR Cl− channels to decrease airway surface liquid volume indicate a novel therapeutic action of the glucocorticoid to reverse airway flooding.This brief review delves into the early non-genomic and late genomic signaling mechanisms of dexamethasone regulation of ion channels and airway surface liquid dynamics, shedding light on the molecular mechanisms underpinning the action of the glucocorticoid in managing COVID-19.

The COPD-Associated Polymorphism Impairs the CFTR Function to Suppress Excessive IL-8 Production upon Environmental Pathogen Exposure.

COPD is a lifestyle-related disease resulting from irreversible damage to respiratory tissues mostly due to chronic exposure to environmental pollutants, including cigarette smoke. Environmental pathogens and pollutants induce the acquired dysfunction of the CFTR Cl- channel, which is invoked in COPD. Despite the increased incidence of CFTR polymorphism R75Q or M470V in COPD patients, the mechanism of how the CFTR variant affects COPD pathogenesis remains unclear. Here, we investigated the impact of CFTR polymorphisms (R75Q, M470V) on the CFTR function in airway epithelial cell models. While wild-type (WT) CFTR suppressed the proinflammatory cytokine production induced by COPD-related pathogens including pyocyanin (PYO), R75Q- or M470V-CFTR failed. Mechanistically, the R75Q- or M470V-CFTR fractional PM activity (FPMA) was significantly lower than WT-CFTR in the presence of PYO. Notably, the CF drug Trikafta corrected the PM expression of R75Q- or M470V-CFTR even upon PYO exposure and consequently suppressed the excessive IL-8 production. These results suggest that R75Q or M470V polymorphism impairs the CFTR function to suppress the excessive proinflammatory response to environmental pathogens associated with COPD. Moreover, Trikafta may be useful to prevent the COPD pathogenesis associated with acquired CFTR dysfunction.

Evaluation of chronic cigarette smoke exposure in human bronchial epithelial cultures.

Cigarette smoke (CS) exposure induces both cytotoxicity and inflammation, and often causes COPD, a growing cause of morbidity and mortality. CS also inhibits the CFTR Cl- channel, leading to airway surface liquid dehydration, which is predicated to impair clearance of inhaled pathogens and toxicants. Numerous in vitro studies have been performed that utilize acute (≤24 h) CS exposures. However, CS exposure is typically chronic. We evaluated the feasibility of using British-American Tobacco (BAT)-designed CS exposure chambers for chronically exposing human bronchial epithelial cultures (HBECs) to CS. HBECs are polarized and contain mucosal and serosal sides. In vivo, inhaled CS interacts with mucosal membranes, and BAT chambers are designed to direct CS to HBEC mucosal surfaces while keeping CS away from serosal surfaces via a perfusion system. We found that serosal perfusion was absolutely required to maintain HBEC viability over time following chronic CS exposure. Indeed, with this system, we found that CS increased inflammation and mucin levels, while decreasing CFTR function. Without this serosal perfusion, CS was extremely toxic within 24 h. We therefore propose that 5- and 10-day CS exposures with serosal perfusion are suitable for measuring chronic CS exposure and can be used for monitoring new and emerging tobacco products.

Select Amino Acid Formulation Influenced Anion Secretion Induced by Th2 Cytokines in Human Bronchial Epithelial Cells (HBECs)

IntroductionAirway surface liquid is tightly regulated by ion channels and/or transporters, and plays a critical role in inflammatory airway diseases, such as ARDS, and in edema fluid clearance during lung injury. Although beta‐adrenergic agonists have been shown to improve alveolar fluid clearance, they have limited effect in late stages of ARDS due to reduced epithelial beta‐adrenergic receptor function or agonist delivery to target regions. CFTR, CLCA2, TMEM16A, and alternative Cl‐/HCO3‐ exchangers (e.g., SLC4 family) are known to be expressed in lung epithelia. Their relative contribution during late stages of lung injury where TH2 cytokines such as IL13 are elevated is not fully understood. The expression pattern of these anion channels are dysregulated in ARDS, and restoration of channel activities could improve fluid accumulation within the lung. In the present study, various anion channel and/or exchanger activities were measured in HBECs exposed to IL13. We have shown that select amino acids influence anion channel activity. Therefore, the effect of a proprietary amino acid formulation on specific anion channels were studied in IL13‐treated HBECs.MethodsTransepithelial short‐circuit current (Isc) was sequentially blocked using inhibitors for ENaC (benzamil), CFTR CFTRInh172), TMEM16A (CaCCInhA01) and Na+/K+/2Cl‐ co‐transporter (bumetanide) in differentiated HBECs mounted in Ussing chambers. Patch‐clamp whole‐cell recordings were performed to determine anion channel activity after IL13 treatment, and channel and/or exchanger expression was analyzed by immunofluorescence.ResultsIL13 increased benzamil‐insensitive (anion) Isc (87.5 ± 4.7 µA.cm‐2 vs 21.9 ± 1.8 µA.cm‐2; P <0.001) and decreased CFTR Isc (1.4 ± 0.52 µA.cm‐2 vs 12.2 ± 0.83 µA.cm‐2; P <0.001) compared to controls. Amino acids reduced benzamil‐insensitive Isc (55.9 ± 4.8 µA.cm‐2; P <0.001) and increased CFTR Isc (6.7 ± 0.75 µA.cm‐2; P <0.001). There was no change in TMEM16A Isc with IL13 treatment. Bumetanide‐sensitive Isc was higher in IL13‐treated cells compared to controls (52.5 ± 3.6 µA.cm‐2 vs 5.5 ± 0.62 µA.cm‐2; P <0.001) suggesting anion secretion via alternate mechanisms, and amino acids decreased bumetanide‐sensitive anion secretion (20.5 ± 2.9 µA.cm‐2; P <0.001). Similarly, whole‐cell patch clamp recordings showed that amino acids restored the CFTR response in IL13‐treated cells. Immunofluorescence imaging demonstrated decreased CFTR expression with IL13, while amino acids recovered CFTR at the apical membrane.ConclusionTreatment with IL13 increased anion secretion while reducing CFTR channel activity in HBECs. Amino acid formulation restored CFTR activity and decreased total anion current in the presence of IL13. These studies suggest anion secretion via alternate anion secretory pathways. Future studies should be directed at understanding the link between CFTR and alternate anion channels in conditions associated with elevated IL13, and how amino acids regulate these channel and/or exchanger functions

Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis.

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl- channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.

Bicarbonate transport of airway surface epithelia in luminally perfused mice bronchioles

HCO3− secretion in distal airways is critical for airway mucosal defense. HCO3−/H+ transport across the apical membrane of airway surface epithelial cells was studied by measuring intracellular pH in luminally microperfused freshly dissected mice bronchioles. Functional studies demonstrated that CFTR, ENaC, Cl−–HCO3− exchange, Na+-H+ exchange, and Na+–HCO3− cotransport are involved in apical HCO3−/H+ transport. RT-PCR of isolated bronchioles detected fragments from Cftr, α, β, γ subunits of ENaC, Ae2, Ae3, NBCe1, NBCe2, NBCn1, NDCBE, NBCn2, Nhe1, Nhe2, Nhe4, Nhe5, Slc26a4, Slc26a6, and Slc26a9. We assume that continuous decline of intracellular pH following alkaline load demonstrates time course of HCO3− secretion into the lumen which is perfused with a HCO3−-free solution. Forskolin-stimulated HCO3− secretion was substantially inhibited by luminal application of CFTRinh-172 (5 μM), H2DIDS (200 μM), and amiloride (1 μM). In bronchioles from a cystic fibrosis mouse model, basal and acetylcholine-stimulated HCO3− secretion was substantially impaired, but forskolin transiently accelerated HCO3− secretion of which the magnitude was comparable to wild-type bronchioles. In conclusion, we have characterized apical HCO3−/H+ transport in native bronchioles. We have demonstrated that cAMP-mediated and Ca2+-mediated pathways are involved in HCO3− secretion and that apical HCO3− secretion is largely mediated by CFTR and H2DIDS-sensitive Cl−–HCO3− exchanger, most likely Slc26a9. The impairment of HCO3− secretion in bronchioles from a cystic fibrosis mouse model may be related to the pathogenesis of early lung disease in cystic fibrosis.

P. aeruginosa Induced Lipid Peroxidation Causes Ferroptotic Cell Death in Airways.

Oxidative stress and infections by Pseudomonas aeruginosa (P. aeruginosa) are prominent in lungs of patients suffering from cystic fibrosis (CF). The present study examines effects of P. aeruginosa on lipid peroxidation in human and mouse lungs, and cell death induced by P. aeruginosa in human airway epithelial cells. The role of the Ca2+ activated Cl- channel TMEM16A, the phospholipid scramblase TMEM16F, and the CFTR Cl- channel for ferroptotic cell death is examined. Lipid peroxidation was detected in human CF lungs, which correlated with bacterial infection. In vivo inoculation with P. aeruginosa or Staphylococcus aureus (S. aureus) induced lipid peroxidation in lungs of mice lacking expression of CFTR, and in lungs of wild type animals. Incubation of CFBE human airway epithelial cells with P. aeruginosa induced an increase in reactive oxygen species (ROS), causing lipid peroxidation and cell death independent of expression of wt-CFTR or F508del-CFTR. Knockdown of TMEM16A attenuated P. aeruginosa induced cell death. Antioxidants such as coenzyme Q10 and idebenone as well as the inhibitor of ferroptosis, ferrostatin-1, inhibited P. aeruginosa-induced cell death. CFBE cells expressing wtCFTR, but not F508del-CFTR, activated a basal Cl- conductance upon exposure to P. aeruginosa, which was caused by an increase in intracellular basal Ca2+ concentrations and activation of Ca2+-dependent adenylate cyclase. The data suggest an intrinsic pro-inflammatory phenotype in CF epithelial cells, while ferroptosis is observed in both non-CF and CF epithelial cells upon infection with P. aeruginosa. CF cells fail to activate fluid secretion in response to infection with P. aeruginosa. The use of antioxidants and inhibitors of ferroptosis is proposed as a treatment of pneumonia caused by infection with P. aeruginosa.

A fungus-derived purpactin A as an inhibitor of TMEM16A chloride channels and mucin secretion in airway epithelial cells

TMEM16A is a Ca2+-activated Cl- channel involved in mucus secretion in inflamed airways and proposed as a drug target for diseases associated with mucus hypersecretion including asthma. This study aimed to identify novel inhibitors of TMEM16A-mediated Cl- secretion in airway epithelial cells from a collection of compounds isolated from fungi indigenous in Thailand and examine its potential utility in mitigating airway mucus secretion using Calu-3 cells as a study model. Screening of > 400 fungal metabolites revealed purpactin A isolated from a soil-derived fungus Penicillium aculeatum PSU-RSPG105 as an inhibitor of TMEM16A-mediated Cl- transport with an IC50 value of ~2 µM. A consistent inhibitory effect of purpactin A on TMEM16A were observed regardless of TMEM16A activators or in the presence of an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a negative regulator of TMEM16A. In addition, purpactin A did not affect cell viability, epithelial barrier integrity and activities of membrane transport proteins essential for maintaining airway hydration including CFTR Cl- channels and apical BK K+ channels. Intriguingly, purpactin A prevented a Ca2+-induced mucin release in cytokine-treated airway cells. Taken together, purpactin A represents the first class of TMEM16A inhibitor derived from fungus, which may be beneficial for the treatment of diseases associated with mucus hypersecretion.

Repeat Dosing of AAV2.5T to Ferret Lungs Elicits an Antibody Response That Diminishes Transduction in an Age-Dependent Manner

Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing-antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures and complemented CFTR Cl– currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200%–300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat dosing significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.

Informatics-guided drug repurposing for Autosomal Dominant Polycystic Kidney Disease (ADPKD).

Informatics-guided drug repurposing for Autosomal Dominant Polycystic Kidney Disease (ADPKD).

TMEM16A chloride channel does not drive mucus production.

Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.

Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4stimulation leads to CFTR channel activation. TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

Role of ion channels in gastrointestinal cancer.

In their seminal papers Hanahan and Weinberg described oncogenic processes a normal cell undergoes to be transformed into a cancer cell. The functions of ion channels in the gastrointestinal (GI) tract influence a variety of cellular processes, many of which overlap with these hallmarks of cancer. In this review we focus on the roles of the calcium (Ca2+), sodium (Na+), potassium (K+), chloride (Cl-) and zinc (Zn2+) transporters in GI cancer, with a special emphasis on the roles of the KCNQ1 K+ channel and CFTR Cl- channel in colorectal cancer (CRC). Ca2+ is a ubiquitous second messenger, serving as a signaling molecule for a variety of cellular processes such as control of the cell cycle, apoptosis, and migration. Various members of the TRP superfamily, including TRPM8, TRPM7, TRPM6 and TRPM2, have been implicated in GI cancers, especially through overexpression in pancreatic adenocarcinomas and down-regulation in colon cancer. Voltage-gated sodium channels (VGSCs) are classically associated with the initiation and conduction of action potentials in electrically excitable cells such as neurons and muscle cells. The VGSC NaV1.5 is abundantly expressed in human colorectal CRC cell lines as well as being highly expressed in primary CRC samples. Studies have demonstrated that conductance through NaV1.5 contributes significantly to CRC cell invasiveness and cancer progression. Zn2+ transporters of the ZIP/SLC39A and ZnT/SLC30A families are dysregulated in all major GI organ cancers, in particular, ZIP4 up-regulation in pancreatic cancer (PC). More than 70 K+ channel genes, clustered in four families, are found expressed in the GI tract, where they regulate a range of cellular processes, including gastrin secretion in the stomach and anion secretion and fluid balance in the intestinal tract. Several distinct types of K+ channels are found dysregulated in the GI tract. Notable are hERG1 upregulation in PC, gastric cancer (GC) and CRC, leading to enhanced cancer angiogenesis and invasion, and KCNQ1 down-regulation in CRC, where KCNQ1 expression is associated with enhanced disease-free survival in stage II, III, and IV disease. Cl- channels are critical for a range of cellular and tissue processes in the GI tract, especially fluid balance in the colon. Most notable is CFTR, whose deficiency leads to mucus blockage, microbial dysbiosis and inflammation in the intestinal tract. CFTR is a tumor suppressor in several GI cancers. Cystic fibrosis patients are at a significant risk for CRC and low levels of CFTR expression are associated with poor overall disease-free survival in sporadic CRC. Two other classes of chloride channels that are dysregulated in GI cancers are the chloride intracellular channels (CLIC1, 3 & 4) and the chloride channel accessory proteins (CLCA1,2,4). CLIC1 & 4 are upregulated in PC, GC, gallbladder cancer, and CRC, while the CLCA proteins have been reported to be down-regulated in CRC. In summary, it is clear, from the diverse influences of ion channels, that their aberrant expression and/or activity can contribute to malignant transformation and tumor progression. Further, because ion channels are often localized to the plasma membrane and subject to multiple layers of regulation, they represent promising clinical targets for therapeutic intervention including the repurposing of current drugs.

Discovery of a novel chalcone derivative inhibiting CFTR chloride channel via AMPK activation and its anti-diarrheal application

Secretory diarrhea is one of the most common causes of death world-wide especially in children under 5 years old. Isoliquiritigenin (ISLQ), a plant-derived chalcone, has previously been shown to exert anti-secretory action in vitro and in vivo by inhibiting CFTR Cl− channels. However, its CFTR inhibition potency is considerably low (IC50 > 10 μM) with unknown mechanism of action. This study aimed to identify novel chalcone derivatives with improved potency and explore their mechanism of action. Screening of 27 chalcone derivatives identified CHAL-025 as the most potent chalcone analog that reversibly inhibited CFTR-mediated Cl− secretion in T84 cells with an IC50 of ∼1.5 μM. As analyzed by electrophysiological and biochemical analyses, the mechanism of CFTR inhibition by CHAL-025 is through AMP-activated protein kinase (AMPK), a negative regulator of CFTR activity. Furthermore, Western blot analyses and molecular dynamics (MD) results suggest that CHAL-025 activates AMPK by binding at the allosteric site of an upstream kinase calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, CHAL-025 inhibited both cholera toxin (CT) and bile acid-induced Cl− secretion in T84 cells and prevented CT-induced intestinal fluid secretion in mice. Therefore, CHAL-025 represents a promising anti-diarrheal agent that inhibits CFTR Cl− channel activity via CaMKKβ-AMPK pathways.

The function of the CFTR Cl- channel in the exocrine/endocrine pancreas under pathological conditions

We present simulations for electron transmission through a platinum macrocapillary (diameter d = 3.3 mm, length l = 48 mm) using classical transport theory. Both elastic and inelastic scattering events of primary electrons colliding with the inner wall of the capillary are taken into account. We also model the generation and transport of secondary electrons inside the material. We find excellent agreement of our simulated electron-energy spectra with recent experimental data for 200 eV primary electrons.

Loss of CFTR Function Increases Wnt/Planar Cell Polarity (PCP) Signaling and Tight Junction Permeability in Intestinal Epithelium

Cystic fibrosis (CF) patients and cystic fibrosis transmembrane conductance regulator (Cftr) knockout (KO) mice exhibit increased intestinal paracellular permeability. This can lead to the translocation of bacterial toxins from the gut lumen into the circulation, adversely affecting liver and lung function. However, the molecular mechanism(s) underlying increased paracellular permeability in CF is unknown. Loss of Cftr Cl− and HCO3− channel function increased intracellular pH (pHi) in crypt epithelium, causing intestinal stem cell hyperproliferation via increased Wnt/β‐catenin signaling. An alkaline pHi increases plasma membrane binding of the Wnt transducer Dishevelled (Dvl), which is postulated to stabilize the association of Dvl with the Wnt receptor Frizzled. We hypothesized that this mechanism would also increase Dvl‐mediated transduction of Wnt/PCP signaling, which regulates cell migration, cell polarity and tight junction (TJ) remodeling. In this study, Dvl2 apical membrane association was measured by fluorescence confocal microscopy in transit‐amplifying cells of enteroid crypts from wild‐type (WT) and Cftr KO mice expressing Dvl2‐EGFP and membrane Tomato (RosamT/mG). Immunofluorescence was used to assess TJ localization of the Wnt/PCP receptor Frizzled 6 (Fz6) and the Rho GTPase Cdc42, a downstream target of Wnt/PCP signaling that regulates intestinal polarity and migration. Paracellular permeability was estimated as the influx rate of Cascade Blue®‐labeled dextran into enteroid lumens using confocal microscopy. Western blot analysis compared Cdc42 protein expression and active (GTP‐bound) Cdc42 was measured via a colorimetric assay in WT and CFTR KO Caco2 cell lines. Quantitative RT‐PCR was used in evaluating the mRNA expression of multiple TJ‐associated proteins in freshly isolated murine crypts. Cftr KO enteroids had increased paracellular permeability relative to WT. Dvl2‐EGFP apical membrane association was increased ~2‐fold in Cftr KO enteroid crypts, as compared to that in WT, and co‐localized with Fz6. Cdc42 was increased in TJ localization, protein expression and activation in CF intestinal epithelium, as compared to WT. mRNA expression of Par3 and Par6b, which are involved in the remodeling of TJ proteins, was significantly decreased in Cftr KO crypts, as compared to WT. In contrast, mRNA expression in Cftr KO crypts of TJ structural protein claudin‐2 was increased, which correlates with greater epithelial Na+ permeability. These findings are consistent with the hypothesis that the alkaline pHi of CF crypt epithelium increases Dvl‐dependent transduction of Wnt/PCP signaling, leading to increased paracellular permeability via TJ remodeling. Wnt/PCP signaling may be a potential target to normalize intestinal paracellular permeability in CF patients.Support or Funding InformationSupported by NIH DK048816 and the Cystic Fibrosis Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tobramycin reduces key virulence determinants in the proteome of Pseudomonas aeruginosa outer membrane vesicles

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.

Cyclodextrins reduce the ability of Pseudomonas aeruginosa outer-membrane vesicles to reduce CFTR Cl- secretion.

Pseudomonas aeruginosa secretes outer-membrane vesicles (OMVs) that fuse with cholesterol-rich lipid rafts in the apical membrane of airway epithelial cells and decrease wt-CFTR Cl- secretion. Herein, we tested the hypothesis that a reduction of the cholesterol content of CF human airway epithelial cells by cyclodextrins reduces the inhibitory effect of OMVs on VX-809 (lumacaftor)-stimulated Phe508del CFTR Cl- secretion. Primary CF bronchial epithelial cells and CFBE cells were treated with vehicle, hydroxypropyl-β-cyclodextrin (HPβCD), or methyl-β-cyclodextrin (MβCD), and the effects of OMVs secreted by P. aeruginosa on VX-809 stimulated Phe508del CFTR Cl- secretion were measured in Ussing chambers. Neither HPβCD nor MβCD were cytotoxic, and neither altered Phe508del CFTR Cl- secretion. Both cyclodextrins reduced OMV inhibition of VX-809-stimulated Phe508del-CFTR Cl- secretion when added to the apical side of CF monolayers. Both cyclodextrins also reduced the ability of P. aeruginosa to form biofilms and suppressed planktonic growth of P. aeruginosa. Our data suggest that HPβCD, which is in clinical trials for Niemann-Pick Type C disease, and MβCD, which has been approved by the U.S. Food and Drug Administration for use in solubilizing lipophilic drugs, may enhance the clinical efficacy of VX-809 in CF patients when added to the apical side of airway epithelial cells, and reduce planktonic growth and biofilm formation by P. aeruginosa. Both effects would be beneficial to CF patients.