A Cl channel in Ascaris suum selectivity conducts dicarboxylic anion products of glucose fermentation and suggests a role in removal of waste organic anions

The permeability of organic anions (produced from anaerobic fermentation of glucose) through a nonselective membrane Cl channel was examined. Single channel recording techniques were used to study the permeabilities of the anions: oxalate, succinate, oxaloacetate, malate, lactate and pyruvate in Ascaris muscle cell membranes. All of the anions, except malate, were found to be conducted through the channel. The relative permeability of most anions could be predicted from the component structure of the anions. The failure of the channel to conduct malate prevents an energy drain on the cell. These studies further the hypothesis that a Cl channel functions to transport waste organic anions across the cell membrane. This mechanism does not require specific exchange carriers for the anions. Channels with properties like the nonselective anion channels of Ascaris, are suitable for transport of carboxylic anions through cell membranes, down a concentration or potential gradient.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.).     

A patch-clamp study of the ionic selectivity of the large conductance,ca-activated chloride channel in muscle vesicles prepared fromAscaris suum

Summary Plasma membrane vesicles prepared from the bag re gion of the somatic muscle cell of the parasiteAscaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of anions has been investigated using the patch-clamp technique on isolated inside-out patches of muscle membrane. Symmetrical Cl solutions (140 mm) produced single-channel I/V plots with reversal potentials of 0 mV, substitution of bath Cl by 140 mM NO₃, Br and I caused depolarizing shifts in the reversal potentials. Replacement of the internal Cl by F (140 mM) caused a large hyperpolarizing shift in the reversal potential. The channel dis played a permeability sequence of I > Br = NO₃> Cl > F which differed from the corresponding conductance sequence Cl > NO₃ = Br = I > F. The ionic environment within the channel pore has been investigated using Reuter and Stevens (1980) plots to describe the selectivity and “fluidity” of the channel pore. In addition, the approach of Wright and Diamond (1977) was employed to estimate the number of cationic binding sites within the channel pore. The channel is relatively fluid but the number of cationic binding sites varies inversely with the ionic radius of the anion from 2.15 for F to 0.89 for the large planar anion NO₃     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda)

Summary A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37–39° C under anaerobic conditions (95% N₂, 5% CO₂). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5–15 min, is totally inhibited at 4° C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22–9° C. Activation also requires favorable pH (6.8–8.7) and continual exposure to sufficiently high sodium concentrations (134–154mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 g/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.Adult Ascaris suum were provided through the generosity of Wilson and Company, Cedar Rapids, Iowa, U.SA. This study was supported by grant number 5T01 HD00152 and postdoctoral fellowship 1F 32AI05646 from the National Institute of Health, U.S.A.     

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H₂ as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H₂ as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high K _m (43 mM) for DMSO. The electron transport from H₂ to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N₂O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - DMS Dimethylsulphide (CH₃)₂S - DMSO Dimethylsulphoxide (CH₃)₂SO - HQNO 2-(Heptyl)-4-hydroxyquinoline-N-oxide - TMAO Trimethylamine-N-oxide - Y _s Growth yield for substrate S     

Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamide-related peptides

Caenorhabditis elegans possesses 22 FMRFamide-like peptide (flp) genes predicted to encode 60 different FMRFamide-related peptides with a range of C-terminal signatures. Peptides from five flp genes (1, 6, 8, 9 and 14) are known to modulate the ovijector of Ascaris suum in vitro. This study examines the physiological effects of peptides from the remaining 17 flp genes such that the variety of FMRFamide-related peptide-induced ovijector response types can be delineated. Five categories of response were identified according to the pattern of changes in contractile behaviour and baseline tension. Peptides encoded on 16 flp genes (1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 20) had qualitatively similar inhibitory (response type 1) actions, with the lowest activity thresholds (1 nM) recorded for peptides with FIRFamide or FLRFamide C-terminal signatures. Peptides encoded on four flp genes (2, 18, 19 and 21), and on the A. suum afp-1 gene, had excitatory actions on the ovijector (response type 2), with PGVLRFamides having the lowest activity threshold (1 nM). An flp-2 peptide (LRGEPIRFamide) induced a transient contraction of the ovijector (activity threshold, 10nM) that was designated response type 3. Response type 4 comprised a transient contraction followed by an extended period of inactivity and was observed with peptides encoded on flp-5 (AGAKFIRFamide, APKPKFIRFamide), flp-8 (KNEFIRFamide) and flp-22 (SPSAKWMRFamide). SPSAKWMRFamide was the most potent peptide tested with an activity threshold of 0.1 nM. A single peptide (AMRNALVRFamide; activity threshold 0.1 microM), encoded on flp-11, induced response type 5, a shortening of the ovijector coupled with an increase in contraction frequency. Although most flp genes encode structurally related peptides that trigger one of the five ovijector response types, flp-2 and flp-11 co-encode FMRFamide-related peptides that induce distinct responses. Within the ovijector of A. suum FaRPs play a complex role involving at least five receptor subtypes or signalling pathways.     

The mitochondrial ribosomal RNA genes of the nematodes Caenorhabditis elegans and Ascaris suum: Consensus secondary-structure models and conserved nucleotide sets for phylogenetic analysis

The small- and large-subunit mitochondrial ribosomal RNA genes (mt-s-rRNA and mt-1-rRNA) of the nematode worms Caenorhabditis elegans and Ascaris suum encode the smallest rRNAs so far reported for metazoa. These size reductions correlate with the previously described, smaller, structurally anomalous mt-tRNAs of C. elegans and A. suum. Using primer extension analysis, the 5 end nucleotides of the mt-s-rRNA and mt-1-rRNA genes were determined to be adjacent to the 3 end nucleotides of the tRNA^Glu and tRNA^His genes, respectively. Detailed, consensus secondary-structure models were constructed for the mt-s-rRNA genes and the 3 64% of mt-1-rRNA genes of the two nematodes. The mt-s-rRNA secondary-structure model bears a remarkable resemblance to the previously defined universal core structure of E. coli 16S rRNA: most of the nucleotides that have been classified as variable or semiconserved in the E. coli model appear to have been eliminated from the C. elegans and A. suum sequences. Also, the secondary structure model constructed for the 3 64% of the mt-1-rRNA is similar to the corresponding portion of the previously defined E. coli 23S rRNA core secondary structure. The proposed C. elegans/A. suum mt-s-rRNA and mt-1-rRNA models include all of the secondary-structure element-forming sequences that in E. coli rRNAs contain nucleotides important for A-site and P-site (but not E-site) interactions with tRNAs. Sets of apparently homologous sequences within the mt-s-rRNA and mt-1-rRNA core structures, derived by alignment of the C. elegans and A. suum mt-rRNAs to the corresponding mt-rRNAs of other eukaryotes, and E. coli rRNAs were used in maximum-likelihood analyses. The patterns of divergence of metazoan phyla obtained show considerable agreement with the most prevalent metazoan divergence patterns derived from more classical, morphological, and developmental data.Correspondence to: D.R. Wolstenholme     

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the mu opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.     

Microevolutionary Patterns and Molecular Markers: The Genetics of Geographic Variation in Ascaris suum

Molecular markers have been used only rarely to characterize the population genetic structure of nematodes. Published studies have suggested that different taxa may show distinct genetic architectures. Isoenzyme and RAPD markers have been used to investigate geographic variation of Ascaris suum at the level of infrapopulations (nematodes within individual hosts), within localities, and among geographic regions. Independent estimates of genetic differentiation among population samples based on isoenzyme and RAPD data showed similar patterns and substantial correlation. Heterozygote deficiencies within infrapopulations and large values for inbreeding coefficients among infrapopulations suggested that the composition of these populations was not consistent with a model of random recruitment from a large panmictic pool of life-cycle stages. Both isoenzyme and RAPD markers revealed moderate levels of genetic differentiation among samples representing infrapopulations and localities. Of total gene diversity, 9.4% (isoenzyme) and 9.2% (RAPD) was partitioned among infrapopulations. Geographic localities accounted for 7.8% (isoenzyme) and 6.2% (RAPD) of total diversity. Only infrapopulations from the same farm had low levels of differentiation.     

The distribution of Concanavalin A binding sites on the intestinal epithelium of the nematodes Ascans suum and Parascaris equorum

Summary Receptors for Concanavalin A (Con A), were localized on the intestinal epithelium of the nematodes Ascaris suum and Parascaris equorum. Fixed tissue incubated in ³H-Con A showed labeling of the microvilli surface and basal membrane. Using Con A coupled with peroxidase, the tips of the microvilli of Ascaris suum and the tips and lateral surfaces of Parascaris equorum were stained. The basal membrane of both species was also labeled. No labeling was observed on control tissue incubated without Con A or on tissue incubated with Con A to which -methyl-D-mannoside was added.     

The effect of volatile fatty acids on the inactivation of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> in anaerobic digestion

The application of sludge digestion systems to remove pathogens has been employed to generate biosolids suitable for reuse in agriculture. Traditionally, temperature is considered the principal agent responsible for pathogen reduction in anaerobic digestion. However, other substances such as volatile fatty acids may also have an antimicrobial effect. The objective of this study was to assess the impact of fatty acid mixtures on the inactivation of C. perfringens over a range of digestion temperatures. An equimolar mixture of acetic acid, propionic acid and butyric acid was applied to digester effluent for a period of 24 h at temperatures of 35 °C, 42 °C, 49 °C and 55 °C. C. perfringens inactivation in digester effluents, when dosed with volatile organic acids, was found to depend on pH, acid concentration and temperature. Temperatures above 55 °C appeared to increase the inhibitory effects of the organic acids at higher concentrations. An interaction between temperature and pH on survival of C. perfringens was observed. The results suggest that high concentrations of organic acids at a pH value of 4.5–5.5 during thermophilic digestion substantially reduce concentrations of C. perfringens in municipal sludge.     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

Heterologous expression of Ascaris suum cytochrome b5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli

The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5). It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165-172]. The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.     

Regulation of anaerobic respiratory pathways in Wolinella succinogenes by the presence of electron acceptors

In Wolinella succinogenes ATP synthesis and consequently bacterial growth can be driven by the reduction of either nitrate (E₀=+0.42 V), nitrite (E₀=+0.36 V), fumarate (E₀=+0.03 V) or sulphur (E₀=-0.27 V) with formate as the electron donor. Bacteria growing in the presence of nitrate and fumarate were found to reduce both acceptors simultaneously, while the reduction of both nitrate and fumarate is blocked during growth with sulphur. These observations were paralleled by the presence and absence of the corresponding bacterial reductase activities. Using a specific antiserum, fumarate reductase was shown to be present in bacteria grown with fumarate and nitrate, and to be nearly absent from bacteria grown in the presence of sulphur. The contents of polysulphide reductase, too, corresponded to the enzyme activities found in the bacteria. This suggests that the activities of anaerobic respiration are regulated at the biosynthetic level in W. succinogenes. Thus nitrate and fumarate reduction are repressed by the most electronegative acceptor of anacrobic respiration, sulphur. By contrast, in Escherichia coli a similar effect is exerted by the most electropositive acceptor, O₂. W. succinogenes also differs from E. coli in that fumarate reductase is not repressed by nitrate.Abbreviations BV benzyl viologen - DMN 2,3-dimethyl-1,4-naphthoquinone - DMSO dimethylsulfoxide - TMAO trimethylamine-N-oxide     

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Gliding motility, ultrastructure and nutrition of two newly isolated filamentous sulfate-reducing bacteria, strains 5ac10 and 4be13, were investigated. The filaments were always attached to surfaces. Growth was supported by addition of insoluble aluminium phosphate or agar as substrata for gliding movement. Electron microscopy of ultrathin sections revealed cell walls characteristic of Gramnegative bacteria; the undulated structure of the outer membrane may pertain to the translocation mechanism. Intracytoplasmic membranes were present. Acetate, higher fatty acids, succinate or fumarate served as electron donors and carbon sources. Strain 5ac10 grew also with lactate, but not with benzoate that was used only by strain 4be13. Strain 5ac10 was able to grow slowly on H₂ plus CO₂ or formate in the presence of sulfate without additional organic carbon source. The capacity of complete oxidation was shown by stoichiometric measurements with acetate plus sulfate. Both strains contained b- and c-type cytochromes. Desulfoviridin was detected only in strain 5ac10. The two filamentous gliding sulfate reducers are described as new species of a new genus, Desulfonema limicola and Desulfonema magnum.     

Outwardly rectifying chloride channels and CF: A divorce and remarriage

Outwardly rectifying Cl^– channels were originally thought to be the central element in cystic fibrosis. The role of these channels in CF was questioned to such an extent that doubts were rasied about the validity of the original experiments. Recent data reestablishes a role for outwardly rectifying Cl^– channels (ORCC) in CF and suggests that the protein encoded by the CF gene, the cystic fibrosis transmembrane regulator (CFTR), can effect the regulation of more than one channel in the airway. This minireview deals with the rise, fall, and resurrection of the role of outwardly rectifying Cl^– channels in CF.