CFTR pharmacology.

CFTR protein is an ion channel regulated by cAMP-dependent phosphorylation and expressed in many types of epithelial cells. CFTR-mediated chloride and bicarbonate secretion play an important role in the respiratory and gastrointestinal systems. Pharmacological modulators of CFTR represent promising drugs for a variety of diseases. In particular, correctors and potentiators may restore the activity of CFTR in cystic fibrosis patients. Potentiators are also potentially useful to improve mucociliary clearance in patients with chronic obstructive pulmonary disease. On the other hand, CFTR inhibitors may be useful to block fluid and electrolyte loss in secretory diarrhea and slow down the progression of polycystic kidney disease.

In vitro recapitulation of the site-specific editing (to wild-type) of mutant IDS mRNA transcripts, and the characterization of IDS protein translated from the edited mRNAs.

The transfer of genomic information into the primary RNA sequence can be altered by RNA editing. We have previously shown that genomic variants can be RNA-edited to wild-type. The presence of distinct "edited" iduronate 2-sulfatase (IDS) mRNA transcripts ex vivo evidenced the correction of a nonsense and frameshift variant, respectively, in three unrelated Hunter syndrome patients. This phenomenon was confirmed in various patient samples by a variety of techniques, and was quantified by single-nucleotide primer extension. Western blotting also confirmed the presence of IDS protein similar in size to the wild-type. Since preliminary experimental evidence suggested that the "corrected" IDS proteins produced by the patients were similar in molecular weight and net charge to their wild-type counterparts, an in vitro system employing different cell types was established to recapitulate the site-specific editing of IDS RNA (uridine to cytidine conversion and uridine deletion), and to confirm the findings previously observed ex vivo in the three patients. In addition, confocal microscopy and flow cytometry analyses demonstrated the expression and lysosomal localization in HEK293 cells of GFP-labeled proteins translated from edited IDS mRNAs. Confocal high-content analysis of the two patients' cells expressing wild-type or mutated IDS confirmed lysosomal localization and showed no accumulation in the Golgi or early endosomes.

Structure of wild type and mutant F508del CFTR: A small-angle X-ray scattering study of the protein-detergent complexes.

CFTR is an anionic channel expressed in epithelia whose mutations cause cystic fibrosis. Wild (WT) and mutated (F508del) types were over-expressed in yeast, solubilised in the detergent LPG-14 and purified. The detergent-CFTR complexes were studied by SAXS techniques using a solvent of variable density. The final result of the study is the numerical value of a set of parameters: molecular mass, volume and radius of gyration, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of CFTR and of the detergent are displaced relative to each other. The analysis of these parameters led to the determination of the size and shape of the volumes occupied by protein and by detergent in the complex. WT-CFTR to be an elongated molecule (maximum diameter ∼12.4nm) which spans a flat detergent micelle. The distance distribution function, P(r) confirms that the WT-CFTR is elongated and with an inhomogeneous electronic density. The F508del-CFTR molecule is also elongated (maximum diameter ∼13.2nm), but the associated detergent micelle hides a larger surface, plausibly related to an increased exposure of hydrophobic portions of the mutated protein. The corresponding P(r) is consistent with the presence of well defined domains, probably linked by flexible regions. These differences suggest that the full-length mutant F508del-CFTR has a detectably different conformation, in contrast to the minor differences observed for the isolated F508-containing domain. We interpret the data in terms of an incomplete post-translational assembly of the protein domains.

Functional analysis of acid-activated Cl⁻ channels: properties and mechanisms of regulation.

Cl⁻ channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acid-activated Cl- current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK-293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH5.15), permeability sequence (SCN⁻>I⁻>Br⁻>Cl⁻>gluconate), voltage dependence and sensitivity to blockers of ICl(H) were identical in all cells. These findings suggest a common molecular basis for ICl(H). We analysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR, we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca²âº, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Cl⁻ channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.

Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ΔF508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ΔF508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ΔF508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ΔF508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a direct physical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions.

Direct interaction of a CFTR potentiator and a CFTR corrector with phospholipid bilayers.

Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators and correctors are new drugs that target the basic CFTR protein defect and are expected to benefit cystic fibrosis patients. To optimize the substances so far proposed for human use, and to minimise unwanted side effects, it is essential to investigate possible interactions between the drugs and cell components. We used small-angle X-ray scattering with synchrotron radiation to analyse the effects of two representative drugs, the potentiator VX-770 (Ivacaftor), approved for human use, and the corrector VX-809 (Lumacaftor), on a model phospholipid membrane. By reconstruction of the electron density profile of unilamellar vesicles treated with VX-770 or VX-809 we found that these drugs penetrate the phospholipid bilayer. VX-809 becomes homogeneously distributed throughout the bilayer whereas VX-770 accumulates predominantly in the internal leaflet, behaviour probably favoured by the asymmetry of the bilayer, because of vesicle curvature. Penetration of the bilayer by these drugs, probably as part of the mechanisms of permeation, causes destabilization of the membrane; this must be taken into account during future drug development.

Epithelial sodium channel silencing as a strategy to correct the airway surface fluid deficit in cystic fibrosis.

In the respiratory system, Na(+) absorption and Cl(-) secretion are balanced to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the absence of functional CFTR-dependent Cl(-) secretion. The consequences of defective Cl(-) transport are worsened by the persistence of Na(+) absorption, which contributes to airway surface dehydration. We asked whether normal ASF can be restored to an equal extent by recovering Cl(-) secretion from mutated CFTR or by reducing Na(+) absorption. This is highly relevant in the selection of the best strategy for the treatment of patients with CF. We analyzed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na(+) channel (ENaC) with specific short, interfering RNAs (siRNAs) and after the pharmacological stimulation of CFTR. Our results indicate that (1) single siRNAs complementary to ENaC subunits are sufficient to reduce ENaC transcripts, Na(+) channel activity, and fluid transport, but only silencing both the α and ß ENaC subunits at the same time leads to an increase of ASF (from nearly 7 µm to more than 9 µm); (2) the ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation in non-CF epithelia (10-14 µm); and (3) the pharmacological rescue of mutant CFTR increases the ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of airway hydration in vitro.

Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia.

The TMEM16A protein has a potential role as a Ca(2+)-activated Cl(-) channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o-). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short-interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca(2+)-dependent Cl(-) transport. Following treatment for 24-72 h with interleukin-4 (IL-4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL-4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A-positive cells when epithelia were treated with IL-4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl(-) channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL-4, and that TMEM16A upregulation by IL-4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.

Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations.

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.

A minimal isoform of the TMEM16A protein associated with chloride channel activity.

TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.

Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH.

Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 µA/cm(2) and K(d) = 32.7, 56.6 and 71.9 µm at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 µm at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.

Regulation of TMEM16A chloride channel properties by alternative splicing.

Expression of TMEM16A protein is associated with the activity of Ca(2+)-activated Cl(-) channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca(2+) sensitivity by nearly 4-fold, resulting in Cl(-) currents requiring lower Ca(2+) concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca(2+)-activated Cl(-) channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca(2+) dependence of the TMEM16A-dependent Cl(-) channels in a tissue-specific manner.

Lack of Kcnn4 improves mucociliary clearance in muco-obstructive lung disease.

Airway mucociliary clearance (MCC) is the main mechanism of lung defense keeping airways free of infection and mucus obstruction. Airway surface liquid volume, ciliary beating, and mucus are central for proper MCC and critically regulated by sodium absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive diseases. The calcium-activated potassium channel KCa.3.1, encoded by Kcnn4, participates in ion secretion, and studies showed that its activation increases Na+ absorption in airway epithelia, suggesting that KCa3.1-induced hyperpolarization was sufficient to drive Na+ absorption. However, its role in airway epithelium is not fully understood. We aimed to elucidate the role of KCa3.1 in MCC using a genetically engineered mouse. KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency and MCC ex vivo and in vivo. Kcnn4 silencing in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+ absorption, improved MCC, reduced Na+ absorption, and did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration, and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+ channel modulation may be a therapeutic strategy to treat muco-obstructive lung diseases.

Epithelial sodium channel inhibition in primary human bronchial epithelia by transfected siRNA.

Na(+) absorption and Cl(-) secretion are in equilibrium to maintain an appropriate airway surface fluid volume and ensure appropriate mucociliary clearance. In cystic fibrosis, this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in the absence of functional CFTR protein, which in turn results in deficient cAMP-dependent Cl(-) secretion and predominant Na(+) absorption. It has been suggested that down-regulation of the epithelial sodium channel, ENaC, might help to restore airway hydration and reverse the airway phenotype in patients with cystic fibrosis. We used an siRNA approach to analyze the possibility of down-regulating ENaC function in bronchial epithelia and examine the resulting effects on fluid transport. siRNA sequences complementary to each of the three ENaC subunits have been used to establish whether single subunit down-regulation is enough to reduce Na(+) absorption. Transfection was performed by exposure to siRNA for 24 hours at the time of cell seeding on permeable support. By using primary human bronchial epithelial cells we demonstrate that (1) siRNA sequences complementary to ENaC subunits are able to reduce ENaC transcripts and Na(+) channel activity by 50 to 70%, (2) transepithelial fluid absorption decreases, and (3) these functional effects last at least 8 days. A decrease in ENaC mRNA results in a significant reduction of ENaC protein function and fluid absorption through the bronchial epithelium, indicating that an RNA interference approach may improve the airway hydration status in patients with cystic fibrosis.

Mutation-specific potency and efficacy of cystic fibrosis transmembrane conductance regulator chloride channel potentiators.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The mutations G551D and G1349D, which affect the nucleotide-binding domains (NBDs) of CFTR protein, reduce channel activity. This defect can be corrected pharmacologically by small molecules called potentiators. CF mutations residing in the intracellular loops (ICLs), connecting the transmembrane segments of CFTR, may also reduce channel activity. We have investigated the extent of loss of function caused by ICL mutations and the sensitivity to pharmacological stimulation. We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. The 1,4-dihydropyridine felodipine and the phenylglycine PG-01 [2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide] were strongly effective on the three CFTR mutants. The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Our results indicate that CFTR potentiators correct the basic defect caused by CF mutations residing in different CFTR domains. However, there are differences among potentiators, with felodipine and PG-01 having a wider pharmacological activity, and SF-01 being more mutation specific. Our observations are useful in the prioritization and development of drugs targeting the CF basic defect.

Nanomolar CFTR inhibition by pore-occluding divalent polyethylene glycol-malonic acid hydrazides.

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential application as antisecretory therapy in cholera. We synthesized mono- and divalent CFTR inhibitors consisting of a malonic acid hydrazide (MalH) coupled via a disulfonic stilbene linker to polyethylene glycols (PEGs; 0.2-100 kDa). IC50 values for CFTR inhibition were 10-15 microM for the monovalent MalH-PEGs, but substantially lower for divalent MalH-PEG-MalH compounds, decreasing from 1.5 to 0.3 microM with increasing PEG size and showing positive cooperativity. Whole-cell patch-clamp showed voltage-dependent CFTR block with inward rectification. Outside-out patch-clamp showed shortened single-channel openings, indicating CFTR pore block from the extracellular side. Luminally added MalH-PEG-MalH blocked by >90% cholera toxin-induced fluid secretion in mouse intestinal loops (IC50 approximately 10 pmol/loop), and greatly reduced mortality in a suckling mouse cholera model. These conjugates may provide safe, inexpensive antisecretory therapy.

Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis.

CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.

Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening.

The most common cause of cystic fibrosis (CF) is deletion of phenylalanine 508 (DeltaF508) in the CF transmembrane conductance regulator (CFTR) chloride channel. The DeltaF508 mutation produces defects in folding, stability, and channel gating. To identify small-molecule correctors of defective cellular processing, we assayed iodide flux in DeltaF508-CFTR-transfected epithelial cells using a fluorescent halide indicator. Screening of 150,000 chemically diverse compounds and more than 1,500 analogs of active compounds yielded several classes of DeltaF508-CFTR correctors (aminoarylthiazoles, quinazolinylaminopyrimidinones, and bisaminomethylbithiazoles) with micromolar potency that produced greater apical membrane chloride current than did low-temperature rescue. Correction was seen within 3-6 hours and persisted for more than 12 hours after washout. Functional correction was correlated with plasma membrane expression of complex-glycosylated DeltaF508-CFTR protein. Biochemical studies suggested a mechanism of action involving improved DeltaF508-CFTR folding at the ER and stability at the cell surface. The bisaminomethylbithiazoles corrected DeltaF508-CFTR in DeltaF508/DeltaF508 human bronchial epithelia but did not correct a different temperature-sensitive CFTR mutant (P574H-CFTR) or a dopamine receptor mutant. Small-molecule correctors may be useful in the treatment of CF caused by the DeltaF508 mutation.

Alpha-aminoazaheterocyclic-methylglyoxal adducts do not inhibit cystic fibrosis transmembrane conductance regulator chloride channel activity.

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential applications in the therapy of secretory diarrheas and polycystic kidney disease. In a recent study, several highly polar alpha-aminoazaheterocyclic-methylglyoxal adducts were reported to reversibly inhibit CFTR chloride channel activity with IC50 values in the low picomolar range (J Pharmacol Exp Ther 322:1023-1035, 2007), more than 10,000-fold better than that of thiazolidinone and glycine hydrazide CFTR inhibitors previously identified by high-throughput screening. In this study, we resynthesized and evaluated the alpha-aminoazaheterocyclic-methylglyoxal adducts reported to have high CFTR inhibition potency (compounds 5, 7, and 8). We verified that the reported synthesis procedures produced the target compounds in high yield. However, we found that these compounds did not inhibit CFTR chloride channel function in multiple cell lines at up to 100 microM concentration, using three independent assays of CFTR function including short-circuit current analysis, whole-cell patch-clamp experiments, and yellow fluorescence protein-fluorescence quenching. As positive controls, approximately 100% of CFTR inhibition was found by thiazolidinone and glycine hydrazide CFTR inhibitors. Our data provide direct evidence against CFTR inhibition by alpha-aminoazaheterocyclic-methylglyoxal adducts.

On the measurement of the functional properties of the CFTR.

A number of methods are currently employed to assess the functional properties of CFTR channels and their response to pharmacological potentiators, correction of the defective CFTR trafficking, and vectorial introduction of new proteins. Here we review the most common methods used to assess CFTR channel function. The suitability of each technique to various experimental conditions is discussed.