Cystic Fibrosis Transmembrane Regulator Channel Research Articles

A Comprehensive and Updated Review of MicroRNA Pathogenesis and Gene Expression in Cystic Fibrosis

MicroRNAs (miRNAs) are short (~22 nucleotides) non-coding sequences that regulate gene expression. It has been recently identified that miR-145 regulates cystic fibrosis transmembrane regulator (CFTR) expression, and response to first-generation miRNAs is recognized as major biomedical research in CF. Abnormal miRNA activity causes numerous diseases, thus the critical role of miRNA is to focus on miRNA bindings to target proteins. miRNA classifications depend on their genomic location and gene structure, and they include intergenic and intronic regions. Intronic miRNA location is in the introns of annotated genes. Intergenic and intronic miRNA genes are controlled with their promoters. Some miRNAs negatively regulate the cystic fibrosis transmembrane conductance regulator (CFTR) gene expression (e.g., miR-101) in adult lung cells and have no effect on fetal epithelial cells. MiR-145, miR-223, and miR-494 directly regulate the CFTR gene expression. MiR-509-3p and miR-494 regulate CFTR more strongly than the other miRNAs. MiR-138 enhances CFTR channels on the cell surface of epithelial cells. Polymorphisms in the 3′-UTR region of CFTR decrease the expression of CFTR. Mutations in the CF (CFTR) gene, located on chromosome 7, diminish ion transport. The consequent loss of chloride and bicarbonate secretion and absorption lead to a multi-organ disease called CF marked by mucus inspissation, chronic inflammation, and tissue destruction. The most affected organs are the lung and pancreas, but the liver, bowel, sinuses, and vas deferens, among others, are prominently involved as well. The loss of CFTR also results in an altered miRNA profile that regulates the response to therapy and disease pathogenesis. This review summarizes miRNA alterations in the CF tissue, the role of miRNA in disease pathogenesis, and the therapeutic opportunity for oligotherapeutic manipulation to improve CF outcomes.

ACEing premature codon termination using anticodon-engineered sup-tRNA-based therapy

ACEing premature codon termination using anticodon-engineered sup-tRNA-based therapy

Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs

Nonsense mutations or premature termination codons (PTCs) comprise ∼11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases; however, their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here, we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542XUGA-, R1162XUGA-, and W1282XUGA-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine-encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential standalone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC-containing endogenous genes.

The Effect of Dynasore Upon the Negative Interaction Between ENaC and CFTR Channels in Xenopus laevis Oocytes.

Shroom is a family of related proteins linked to the actin cytoskeleton, and one of them, xShroom1, is constitutively expressed in Xenopus laevis oocytes which is required for the expression of the epithelial sodium channel (ENaC). On the other hand, ENaC and the cystic fibrosis transmembrane regulator (CFTR) are co-expressed in many types of cells with a negative or positive interaction depending on the studied tissues. Here, we measured the amiloride-sensitive ENaC currents (INaamil) and CFTR currents (ICFTR) with voltage clamp techniques in oocytes co-injected with ENaC and/or CFTR and xShroom1 antisense oligonucleotides. The objective was to study the mechanism of regulation of ENaC by CFTR when xShroom1 was suppressed and the endocytic traffic of CFTR was blocked. CFTR activation had a measurable negative effect on ENaC and this activation resulted in a greater inhibition of INaamil than with xShroom1 antisense alone. Our results with Dynasore, a drug that acts as an inhibitor of endocytic pathways, suggest that the changes in INaamil by xShroom1 downregulation were probably due to an increment in channel endocytosis. An opposite effect was observed when ICFTR was measured. Thus, when xShroom1 was downregulated, the ICFTR was larger than in the control experiments and this effect is not observed with Dynasore. A speculative explanation could be that xShroom1 exerts a dual effect on the endocytic traffic of ENaC and CFTR and these actions were canceled with Dynasore. In the presence of Dynasore, no difference in either INaamil or ICFTR was observed when xShroom1 was downregulated.

Adenylate Cyclase 1 Links Calcium Signaling to CFTR-Dependent Cytosolic Chloride Elevations in Chick Amacrine Cells.

The strength and sign of synapses involving ionotropic GABA and glycine receptors are dependent upon the Cl− gradient. We have shown that nitric oxide (NO) elicits the release of Cl− from internal acidic stores in retinal amacrine cells (ACs); temporarily altering the Cl− gradient and the strength or even sign of incoming GABAergic or glycinergic synapses. The underlying mechanism for this effect of NO requires the cystic fibrosis transmembrane regulator (CFTR) but the link between NO and CFTR activation has not been determined. Here, we test the hypothesis that NO-dependent Ca2+ elevations activate the Ca2+-dependent adenylate cyclase 1 (AdC1) leading to activation of protein kinase A (PKA) whose activity is known to open the CFTR channel. Using the reversal potential of GABA-gated currents to monitor cytosolic Cl−, we established the requirement for Ca2+ elevations. Inhibitors of AdC1 suppressed the NO-dependent increases in cytosolic Cl− whereas inhibitors of other AdC subtypes were ineffective suggesting that AdC1 is involved. Inhibition of PKA also suppressed the action of NO. To address the sufficiency of this pathway in linking NO to elevations in cytosolic Cl−, GABA-gated currents were measured under internal and external zero Cl− conditions to isolate the internal Cl− store. Activators of the cAMP pathway were less effective than NO in producing GABA-gated currents. However, coupling the cAMP pathway activators with the release of Ca2+ from stores produced GABA-gated currents indistinguishable from those stimulated with NO. Together, these results demonstrate that cytosolic Ca2+ links NO to the activation of CFTR and the elevation of cytosolic Cl−.

Inhibitory activities of curzerenone, curdione, furanodienone, curcumol and germacrone on Ca2+-activated chloride channels

Calcium-activated chloride channels (CaCCs) as a kind of widely expressed ion channels play crucial roles in a variety of physiological regulation. TMEM16A has been identified as the molecular basis of CaCCs in numerous cell types and is considered a new drug target for many diseases. Regulating the function of TMEM16A through small molecule modulators has become a new strategy to improve respiratory and digestive dysfunction and even tumor therapy. Herein, we obtained 5 sesquiterpenoids, named curzerenone, curdione, furanodienone, curcumol and germacrone with TMEM16A inhibition and revealed their mechanism of action by fluorescent and electrophysiological assays. Cell-based YFP fluorescence data demonstrated that 5 compounds inhibited TMEM16A-mediated I− influx in a dose-dependent manner. To explore the mechanism of 5 compounds on CaCCs, FRT cells with high expression of TMEM16A, HBE, HT-29 and T84 cells and mouse colons were used in short-circuit current assay. Our results showed that 5 compounds inhibited the Ca2+-activated Cl− currents generated by the Eact, ATP and UTP stimulation, and this inhibitory effect was related not only to the direct inhibition of channel opening, but also the inhibition of intracellular Ca2+ concentration and K+ channel activity. In addition to CaCCs, these 5 compounds also had definite inhibitory activities against cystic fibrosis transmembrane regulator (CFTR) at the cellular level. In summary, these compounds have the potential to regulate the activites of TMEM16A/CaCCs and CFTR channels in vitro, providing a new class of lead compounds for the development of drugs for diseases related to chloride channel dysfunction.

Impact of CFTR Modulation on Intestinal pH, Motility, and Clinical Outcomes in Patients With Cystic Fibrosis and the G551D Mutation.

Objectives:A defect in bicarbonate secretion contributes to the pathophysiology of gastrointestinal complications in patients with cystic fibrosis (CF). We measured gastrointestinal pH, clinical outcomes, and intestinal transit profiles in patients with the G551D mutation before and after treatment with ivacaftor, a CF transmembrane regulator channel (CFTR) potentiator.Methods:Observational studies of ivacaftor effectiveness were conducted in the United States and Canada. A subset of subjects ingested a wireless motility capsule (n=10) that measures in vivo pH, both before therapy with ivacaftor and 1 month after treatment; values obtained were compared for mean pH and area under the pH curve, and regional intestinal motility. We also queried subjects about abdominal pain and recorded body weight before and after treatment.Results:One month after administering ivacaftor, a significant increase in mean pH was observed after gastric emptying (P<0.05). Area under the pH curve analyses indicate increased bicarbonate mass (P<0.05 for select 5 min intervals and all segments >30 min); mean weight gain was 1.1 kg (P=0.08). No difference in abdominal pain or regional transit times was seen.Conclusions:CFTR modulation improves the proximal small intestinal pH profile in patients with the G551D CFTR mutation and we observed clinically relevant, contemporaneous weight gain, although it did not reach statistical significance. These data provide in vivo evidence that CFTR is an important regulator of bicarbonate secretion, which may be a translational link between CFTR function and clinical improvement.

Characterization of CFTR Activators and Inhibitors by the use of a Planar Patch Clamp System

Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells where it is involved in the regulation of fluid transport across the epithelium. A large number of mutations in the protein are known to cause CFTR to become dysfunctional and only a few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel (1). CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which then leads to phosphorylation of the channel via protein kinase A (PKA). Screening for CFTR activators is usually done in the absence of forskolin, but in the presence of low concentrations of cAMP. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. For this purpose an alternative approach can be used. In the presence of internal fluoride CFTR is activated as to the same degree as after forskolin activation. We here present data from Nanion's SyncroPatch 384PE where we activate the channel with either forskolin under fluoride free conditions or by internal solution exchange where the fluoride is washed into the intracellular solution. Our results show that the activation of CFTR by either forskolin or intracellular F- is sensitive to glibenclamide and CFTRInh-172 in a dose - and/or voltage dependent manner. Taken together, these experiments show a stable and cost optimized approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR.

CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl(-) conductance.

Proper apical airway surface hydration is essential to maintain lung function. This hydration depends on well‐balanced water resorption and secretion. The mechanisms involved in resorption are still a matter of debate, especially as the measurement of transepithelial water transport remains challenging. In this study, we combined classical short circuit current (ISC) measurements with a novel D2O dilution method to correlate ion and water transport in order to reveal basic transport mechanisms in lung epithelia. D2O dilution method enabled precise analysis of water resorption with an unprecedented resolution. NCI‐H441 cells cultured at an air–liquid interface resorbed water at a rate of 1.5 ± 0.4 μL/(h cm2). Water resorption and ISC were reduced by almost 80% in the presence of the bulk Cl− channel inhibitor 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) or amiloride, a specific inhibitor of epithelial sodium channel (ENaC). However, water resorption and ISC were only moderately affected by forskolin or cystic fibrosis transmembrane regulator (CFTR) channel inhibitors (CFTRinh‐172 and glybenclamide). In line with previous studies, we demonstrate that water resorption depends on ENaC, and CFTR channels have only a minor but probably modulating effect on water resorption. However, the major ENaC‐mediated water resorption depends on an apical non‐CFTR Cl− conductance.

The ΔF508 Gene Mutation of Cystic Fibrosis Transmembrane Regulator Protein Leads to a Progressive Decline of Beta-Cell Function in Mice Carrying This Mutation

Cystic fibrosis (CF) is the most common autosomal-recessive disorder in humans and is due to mutations in the gene encoding for the cystic fibrosis transmembrane regulator (CFTR). In CF patients, the most common mutation is the deletion of Phe508 (Δ F508), which leads to loss of function of CFTR channel. With increased life expectancy of CF patients, several secondary complications occur, such as cystic fibrosis-related diabetes (CFRD), which affects 20% to 40% of CF patients. However, the mechanisms underlying CFRD are unknown. The objective of this study was to examine the impact of the ΔF508 CFTR mutation on glucose homeostasis in mice. To this aim, we compared cftr Δ F508/ Δ F508 (Δ F508) male and female mice to wild-type (WT) littermates. ΔF508 male and female mice had lower body weight compared to WT mice. At 14 weeks of age, both male and female ΔF508 displayed improved oral glucose tolerance as compared to WT animals. This was associated with greater insulin sensitivity and improved beta-cell function in vivo, as assessed by hyperglycemic clamps. In contrast, 6-month-old ΔF508 male mice were insulin resistant and had defective beta-cell function as compared to their WT littermates. In isolated islets from 14-week-old mice, we observed a mild decrease in glucose-induced insulin secretion ex vivo but no difference in the sensitivity to proinflammatory cytokines. These results suggest that the ΔF508 mutation is associated with a subtle impairment in insulin secretion, which becomes apparent when animals become insulin resistant with age.

Targeting protein–protein interactions to rescue Δf508‐cftr: a novel corrector approach to treat cystic fibrosis

Cystic fibrosis (CF) is one of the most prevalent life‐threatening autosomal recessive disorders in the Western World, with an estimated incidence of 1 in 2000–3000 Caucasian newborns and a median age of survival of 40 years. The first report of CF as a distinct pathology was published in 1938 by Dorothy Andersen, a physician at the Babies & Children's Hospital of Columbia University, who differentiated its diagnosis from celiac disease in the pancreas (Andersen, 1938). This seminal description was followed by other contributions that linked CF to altered Cl− permeability in epithelial cells in the gastrointestinal and hepato‐billiary systems, respiratory tract, reproductive system and sweat glands (Kreindler, 2010); and culminated with the discovery of the gene responsible of the disease, referred to as the cystic fibrosis transmembrane regulator (CFTR) (Riordan et al, 1989). The CFTR is a cAMP‐regulated chloride and bicarbonate channel that controls anion conductance in epithelial cells located in mucosal surfaces. Functional alteration of CFTR leads to the accumulation of mucous secretions that cannot be cleared favouring inflammation and infection. Cumulative evidence shows a strong correlation between the severity of the CF phenotype and the degree of CFTR dysfunction, being stronger for those patients where the channel function is absent (Kreindler, 2010). The CF phenotype is caused by more than 1000 mutations of the CFTR gene including missense, such as R117H or G551D that significantly reduce channel activity, and nonsense like G542X, R553X or W1282X, which abrogate protein expression (Kreindler, 2010). However, the most prevalent mutation in CF patients (70%) is the in‐frame deletion of Phenylalanine at position 508 (ΔF508‐CFTR) (Kreindler, 2010; Rowe & Verkman, 2013). This deletion produces a partially unfolded CFTR protein that is retained in the endoplasmic reticulum and diverted to degradation by the proteosome, resulting in a virtually absent CFTR activity …

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.

Biochemical Basis of the Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Immunoglobulin-like Repeats of Filamin

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.

Role of a non‐CFTR chloride channel in intestinal epithelial chloride secretion

The cystic fibrosis transmembrane regulator (CFTR) is the major Cl channel found in the apical membrane of intestinal epithelial cells. Protein kinase A (PKA) phosphorylates and opens CFTR. Exchange protein directly activated by cAMP (Epac) upon binding of cAMP, activates PKA‐independent signaling via small G proteins. We tested the hypothesis that Epac are involved in cAMP‐stimulated Cl secretion. Human intestinal T84 cells and mouse intestine were used for short circuit current (Isc) measurement in response to agonist stimulated Cl secretion. Epac activator, 8‐pCPT‐2′‐O‐Me‐cAMP, elevated [Ca2+]i, activated Rap2 protein and induced Cl secretion in intact and basolateral membrane permeabilized T84 cells and mouse ileum. These effects of 8‐pCPT‐2′‐O‐Me‐cAMP were completely abolished by BAPTA‐AM, but not by H89. In contrast, T84 cells with silenced Epac1 protein had reduced Isc response to forskolin which was completely inhibited by H89 but not by the phospholipase C inhibitor, U73122. The lack of effect of 8‐pCPT‐2′‐O‐Me‐cAMP either to CFTRinhi‐172, glibenclamide or on whole cell patch clamp recording of Cl current in CHO cells transiently expressing the human CFTR, implicates that CFTR channels are not involved. Biophysical characterization of the Epac1‐dependent Cl conductance in Ussing chambers suggests that Epac mediated Cl conductance was hyperpolarization‐activated, inward rectifying, and displays a Cl−&gt;Br−&gt;I− permeability sequence. These results lead us to conclude that the Epac‐Rap‐PLC‐[Ca2+]i signaling pathway is involved in cAMP‐stimulated Cl secretion through non‐CFTR Cl channel.

Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl(-) channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G (j)) and its sensitivity to transjunctional voltage (V (j)) by the dual voltage-clamp method. Application of forskolin induced a Cl(-) current; increased G (j) approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V (j ) gating, monitored by a change in the ratio between G (j) steady state and G (j) peak (G (j)SS/G (j)PK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G (j) and decreased the sensitivity to V (j) gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G (j) and V (j) changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.

Ionic currents in multidrug resistant K562 human leukemic cells

In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 μM), an activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 μM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 μM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 μM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.

Regulation of apical surface fluid and protein secretion in human airway epithelial cell line Calu-3

We evaluated the relationship between apical surface fluid (ASF) and protein secretion in Calu-3 cells grown at an air–liquid interface. Calu-3 monolayers responded to forskolin, a cystic fibrosis transmembrane regulator (CFTR) channel agonist, by secreting a significant amount of ASF. Such a response from Calu-3 monolayers was not observed with CFTR channel blockers glybenclamide and DPC. Other ion channel mediators, PGF-2α, PMA, DNDS, and DIDS, had no effect on Calu-3 ASF secretion. Forskolin decreased Calu-3 protein secretion and glybenclamide increased protein secretion. Similarly, forskolin decreased Calu-3 lysozyme secretion, whereas glybenclamide and DPC increased lysozyme secretion. We observed significant changes in Calu-3 fluid and protein secretions with ion channel mediators known to alter CFTR activity. Our results demonstrate a functional link between fluid and protein secretions in Calu-3 apical surface and suggested a possible involvement of CFTR in these processes.

Tyrosine Kinase c-Src Constitutes a Bridge between Cystic Fibrosis Transmembrane Regulator Channel Failure and MUC1 Overexpression in Cystic Fibrosis

Cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) chloride channel, is associated in the respiratory system with the accumulation of mucus and impaired lung function. The role of the CFTR channel in the regulation of the intracellular pathways that determine the overexpression of mucin genes is unknown. Using differential display, we have observed the differential expression of several mRNAs that may correspond to putative CFTR-dependent genes. One of these mRNAs was further characterized, and it corresponds to the tyrosine kinase c-Src. Additional results suggest that c-Src is a central element in the pathway connecting the CFTR channel with MUC1 overexpression and that the overexpression of mucins is a primary response to CFTR malfunction in cystic fibrosis, which occurs even in the absence of bacterial infection.