Cystic Fibrosis Transmembrane Conductance Regulator Currents Research Articles

Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis.

Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo. Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O2 for 24h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined. Ussing chamber (p<0.05), whole-cell (p<0.001), and single channel patch-clamp (p<0.0001) showed significant inhibition of Cl- currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4h (p<0.0001). Airway surface liquid (p<0.0001), periciliary liquid (p<0.001), and MCT (p<0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p<0.0001), increased HIF1α mRNA (p<0.05), reduced pH (p<0.01), and decreased MCT (p<0.001). SPD measurements demonstrated markedly diminished transepithelial Cl- transport (p<0.0001). Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction.

Mechanistic analysis and significance of sphingomyelinase-mediated decreases in transepithelial CFTR currents in nHBEs.

Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508‐CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib‐1, which also decreased CFTR function. The SMase‐mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5′ adenosine monophosphate‐activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non‐CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508‐CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid‐SMase protein than nHBEs. Lastly, we determined that airway‐like neutrophils, which are increased in the CF lung, secrete acid‐SMase. Identifying the mechanism of SMase‐mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid‐SMase from cell types relevant to CF.

Sphingomyelinase decreases transepithelial anion secretion in airway epithelial cells in part by inhibiting CFTR-mediated apical conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel whose dysfunction causes cystic fibrosis (CF). The loss of CFTR function in pulmonary epithelial cells causes surface dehydration, mucus build‐up, inflammation, and bacterial infections that lead to lung failure. Little has been done to evaluate the effects of lipid perturbation on CFTR activity, despite CFTR residing in the plasma membrane. This work focuses on the acute effects of sphingomyelinase (SMase), a bacterial virulence factor secreted by CF relevant airway bacteria which degrades sphingomyelin into ceramide and phosphocholine, on the electrical circuitry of pulmonary epithelial monolayers. We report that basolateral SMase decreases CFTR‐mediated transepithelial anion secretion in both primary bronchial and tracheal epithelial cells from explant tissue, with current CFTR modulators unable to rescue this effect. Focusing on primary cells, we took a holistic ion homeostasis approach to determine a cause for reduced anion secretion following SMase treatment. Using impedance analysis, we determined that basolateral SMase inhibits apical and basolateral conductance in non‐CF primary cells without affecting paracellular permeability. In CF primary airway cells, correction with clinically relevant CFTR modulators did not prevent SMase‐mediated inhibition of CFTR currents. Furthermore, SMase was found to inhibit only apical conductance in these cells. Future work should determine the mechanism for SMase‐mediated inhibition of CFTR currents, and further explore the clinical relevance of SMase and sphingolipid imbalances.

Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP

We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.

Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells.

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells. CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements. A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells. These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

The G551D CFTR chloride channel spurs the development of personalized medicine.

The G551D CFTR chloride channel spurs the development of personalized medicine.

Cystic fibrosis transmembrane conductance regulator‐mRNA delivery: a novel alternative for cystic fibrosis gene therapy

Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA. We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches. Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection. From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.

Multicenter Intestinal Current Measurements in Rectal Biopsies from CF and Non-CF Subjects to Monitor CFTR Function

Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm2; 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (P<0.005, CF vs non-CF for all parameters). Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98). CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects) were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533). Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects). Multicenter ICM is a feasible CFTR outcome measure that discriminates CF from non-CF ion transport, offers unique advantages over other CFTR bioassays, and warrants further development as a potential CFTR biomarker.

Ethanol and its non-oxidative metabolites profoundly inhibit CFTR function in pancreatic epithelial cells which is prevented by ATP supplementation

Excessive alcohol consumption is a major cause of acute pancreatitis, but the mechanism involved is not well understood. Recent investigations suggest that pancreatic ductal epithelial cells (PDECs) help defend the pancreas from noxious agents such as alcohol. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel plays a major role in PDEC physiology and mutated CFTR is often associated with pancreatitis, we tested the hypothesis that ethanol affects CFTR to impair ductal function. Electrophysiological studies on native PDECs showed that ethanol (10 and 100mM) increased basal, but reversibly blocked, forskolin-stimulated CFTR currents. The inhibitory effect of ethanol was mimicked by its non-oxidative metabolites, palmitoleic acid ethyl ester (POAEE) and palmitoleic acid (POA), but not by the oxidative metabolite, acetaldehyde. Ethanol, POAEE and POA markedly reduced intracellular ATP (ATPi) which was linked to CFTR inhibition since the inhibitory effects were almost completely abolished if ATPi depletion was prevented. We propose that ethanol causes functional damage of CFTR through an ATPi-dependent mechanism, which compromises ductal fluid secretion and likely contributes to the pathogenesis of acute pancreatitis. We suggest that the maintenance of ATPi may represent a therapeutic option in the treatment of the disease.

Increased apical Na+ permeability in cystic fibrosis is supported by a quantitative model of epithelial ion transport

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an anion channel. In the human lung CFTR loss causes abnormal ion transport across airway epithelial cells. As a result CF individuals produce thick mucus, suffer persistent bacterial infections and have a much reduced life expectancy. Trans-epithelial potential difference (Vt) measurements are routinely carried out on nasal epithelia of CF patients in the clinic. CF epithelia exhibit a hyperpolarised basal Vt and a larger Vt change in response to amiloride (a blocker of the epithelial Na+ channel, ENaC). Are these altered bioelectric properties solely a result of electrical coupling between the ENaC and CFTR currents, or are they due to an increased ENaC permeability associated with CFTR loss? To examine these issues we have developed a quantitative mathematical model of human nasal epithelial ion transport. We find that while the loss of CFTR permeability hyperpolarises Vt and also increases amiloride-sensitive Vt, these effects are too small to account for the magnitude of change observed in CF epithelia. Instead, a parallel increase in ENaC permeability is required to adequately fit observed experimental data. Our study provides quantitative predictions for the complex relationships between ionic permeabilities and nasal Vt, giving insights into the physiology of CF disease that have important implications for CF therapy.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

Prostaglandin E2 Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Activity and Airway Surface Liquid Volume Requires Gap Junctional Communication

Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E₂ (PGE₂) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coordination of these pathways for modulating CFTR activity and mucus hydration. We used RT-PCR and Western blotting to determine connexin (Cx), CD73, and EP-R expression in a Calu-3 airway epithelial cell line grown on Transwell (Corning Costar, Cambridge, MA) inserts. We used dye coupling to evaluate gap junctional intercellular communication (GJIC). We used Ussing chamber studies and X-Z confocal microscopy to monitor Cl(-) secretion and ASL volume regulation. We found that connexin 43 (Cx43)-mediated GJIC was increased either by endogenous ADO after the hydrolysis of purine nucleotides by CD73 or by the direct activation of ADO-Rs. Inhibition of phospholipase A2 and cyclooxygenase prevented ADO-dependent increases in GJIC, suggesting the involvement of PGE₂. PGE₂ was found to increase GJIC markedly by stimulating EP4-Rs. The modulation of ADO signaling also affected the PAR-dependent activation of CFTR. The reduction of GJIC by CD73 or Cx43 inhibition prevented PAR-evoked CFTR currents in Ussing chambers. The inhibition of GJIC resulted in a failure of PGE₂ to increase ASL volume in Calu-3 cells and in primary cultures of well-differentiated human airway epithelial cells. Thus, gap junctions coordinate a signaling network comprising CFTR, ADO-Rs, PARs, and EP-Rs, and are required for ASL volume homeostasis.

The cigarette smoke component cadmium inhibits the expression and function of the CFTR chloride channel in lung epithelial cells

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that is mostly expressed in epithelial cells. Mutations in the cftr gene, that affect CFTR trafficking and/or function, lead to Cystic Fibrosis. Cystic Fibrosis is an inherited chronic disease affecting primarily the lungs. Therefore CFTR is necessary for the normal function of this organ. It was recently reported that cigarette smoke reduces the expression of CFTR (Cantin et al., 2006). However, the mechanisms underlying this inhibitory effect were not elucidated. We found that the suppressor effects of cigarette smoke could be specifically reproduced by cadmium, but not other cigarette smoke components such as nicotine (an alkaloid) or acrolein (an aldehyde). The cigarette smoke component cadmium (50 μM) decreases the expression of the CFTR protein by 61.5 ± 10.6 % in the bronchial epithelial cells Calu‐3. Furthermore, Calu‐3 cells treated for 5 days with 50 μM cadmium resulted in 38.9 ± 6.3% inhibition of CFTR currents indicating that CFTR function was also affected by cadmium in polarized cells. Cadmium is a toxic heavy metal that is present in cigarette smoke as well as contaminated air, food and water, and is believed to interfere with several physiological processes. Our findings identify cadmium as a key component present in cigarette smoke that is responsible for the inhibition of CFTR observed in lung epithelial cells.

Discovery of α-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl(-) channels. In whole-cell patch-clamp experiments of Chinese hamster ovary (CHO) cells expressing WT-CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM concentrations. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC(50) = 14.7 microM), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172) (IC(50) = 1.2 microM), and alpha-aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC(50) = 71 pM), 8a,b (IC(50) = 2.5 nM), and 7a,b (IC(50) = 3.4 nM) as the most potent inhibitors of WT-CFTR channels. Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). No effect of compound 5a was detected on the volume-regulated or calcium-regulated iodide efflux. Picomolar inhibition of WT-CFTR with adduct 5a was also found using a 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescent probe applied to the human tracheobronchial epithelial cell line 16HBE14o-. Finally, we found comparable inhibition by 5a or by CFTR(inh)-172 of forskolin-dependent short-circuit currents in mouse colon. To the best of our knowledge, these new nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts represent the most potent compounds reported to inhibit CFTR chloride channels.

Dynamic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Competitive Interactions of Molecular Adaptors

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.

Interregulation of Proton-gated Na+ Channel 3 and Cystic Fibrosis Transmembrane Conductance Regulator

Proton-gated Na(+) channels (ASIC) are new members of the epithelial sodium channel/degenerin gene family. ASIC3 mRNA has been detected in the homogenate of pulmonary tissues. However, whether ASIC3 is expressed in the apical membranes of lung epithelial cells and whether it regulates cystic fibrosis transmembrane conductance regulator (CFTR) function are not known at the present time. Using reverse transcription-PCR, we found that the ASIC3 mRNA was expressed in the human airway mucosal gland (Calu-3) and human airway epithelial (16HBE14o) cells. Indirect immunofluorescence microscopy revealed that ASIC3 was co-segregated with CFTR in the apical membranes of Calu-3 cells. Proton-gated, amiloride-sensitive short circuit Na(+) currents were recorded across Calu-3 monolayers mounted in an Ussing chamber. In whole-cell patch clamp studies, activation of CFTR channels with cAMP reduced proton-gated Na(+) current in Calu-3 cells from -154 +/- 28 to -33 +/- 16 pA (n = 5, p < 0.05) at -100 mV. On the other hand, cAMP-activated CFTR activity was significantly inhibited following constitutive activation of putative ASIC3 at pH 6.0. Immunoassays showed that both ASIC3 and CFTR proteins were expressed and co-immunoprecipitated mutually in Calu-3 cells. Similar results were obtained in human embryonic kidney 293T cells following transient co-transfection of ASIC3 and CFTR. Our results indicate that putative CFTR and ASIC3 channels functionally interact with each other, possibly via an intermolecular association. Because acidic luminal fluid in the cystic fibrosis airway and lung tends to stimulate ASIC3 channel expression and activity, the interaction of ASIC3 and CFTR may contribute to defective salt and fluid transepithelial transport in the cystic fibrotic pulmonary system.

Response to “β-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes”

To the Editor : We read the report of Lin et al. ([1][1]) with great interest. The authors suggest that observed increases in the Ni2+-sensitive bidirectional current after exposure of the mouse, rat, and guinea pig myocytes to isoproterenol were in fact not attributable to changes in the Na+/Ca2+