Transepithelial Chloride Transport Research Articles

Regulation of ClC-K/barttin by endocytosis influences distal convoluted tubule hyperplasia.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

The rs12532734 Polymorphism Near the Solute Carrier 26A3 Gene Locus Is Associated With Gallstone Disease in Children.

Gallstones are increasingly frequent in children. In this candidate gene study, we genotyped 5 gene variants ( ANO1 , SPTLC3 , TMEM147 , TNRC6B , rs12532734) from a recent gallstone genome-wide association study (GWAS) in a cohort of 214 children with gallstones and 172 gallstone-free adult controls. In total, 138 genotyped children presented with symptomatic gallstone disease, 47 underwent cholecystectomy, and 126 received ursodeoxycholic acid (UDCA) as therapy for stones. Among 5 tested variants, the rs12532734 polymorphism modulated the gallstone risk in the studied cohort. Its genotype distribution significantly ( P = 0.025) departed from the Hardy-Weinberg equilibrium among cases, and the common allele was associated with increased odds of developing gallstones at young age (OR = 1.69, P = 0.014). SLC26A3 is the nearest gene to rs12532734 and is involved in the transepithelial bicarbonate and chloride transport. The association of rs12532734 with pediatric gallstones is a novel finding warranting further investigations also with regard to biliary bicarbonate flux and bile composition.

Electro-steric opening of the clc-2 chloride channel gate

The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure–function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.

Effect of apical chloride concentration on the measurement of responses to CFTR modulation in airway epithelia cultured from nasal brushings.

IntroductionOne method for assessing the in vitro response to CFTR‐modulating compounds is by analysis of epithelial monolayers in an Ussing chamber, where the apical and basolateral surfaces are isolated and the potential difference, short‐circuit current, and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined.MethodsCFTR‐mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX‐661, C4/C18, 4‐phenylbutyrate (4‐PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr.ResultsImposing a chloride gradient increased CFTR‐mediated and CaCC‐mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity, which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature‐mediated F508del CFTR correction.ConclusionsImposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR‐mediated ion transport in expanded non‐CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia, the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride, resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy.

125Iodide as a Surrogate for 36Chloride in Tracing Transepithelial Intestinal Chloride Transport

Chloride (Cl−) transport by the large intestine is critically important, driving epithelial fluid secretion and absorption. Dysregulation of Cl− transport by the intestine results in diarrhea‐associated pathologies. Measurement of epithelial Cl− transport has long been achieved using the radioisotope 36Cl−. However, the price of 36Cl has sky‐rocketed, now costing over $100 per μCi compared to just $1.50 per μCi 15 years ago, making it prohibitively expensive and a barrier to future research. 125Iodide (125I−) has been used as an alternative to 36Cl− in some transport assays, including Cl− efflux and influx by cultured cells, but has never before been validated in parallel with 36Cl as an alternative for tracing transepithelial Cl− transport. The goal of this study was to examine whether 125I− can serve as a reliable surrogate for measuring Cl− transport by the intestinal epithelium. Under symmetrical short‐circuited conditions in the Ussing chamber, we simultaneously measured 36Cl− and 125I− fluxes across the cecum and distal colon from mice lacking the main Cl−/HCO3− exchanger, DRA (Slc26a3), relative to wild‐type controls. In addition, we used a cocktail of forskolin and 3‐isobutyl‐1‐methylxanthine (IBMX) to stimulate cAMP production and net Cl− secretion by these tissues. We found the unidirectional fluxes of 125I− correlated well with 36Cl− in all circumstances, even in the absence of DRA, with a significant R2 value of 0.86 across all fluxes, or higher (up to 0.93) when data was separated by genotype and intestinal segment. Unexpectedly, cAMP induced net Cl− secretion predominantly through a reduction in the absorptive, mucosal‐to‐serosal, Cl− flux rather than stimulating the secretory serosal‐to‐mucosal Cl− flux, which is noteworthy considering antidiarrheal drugs often target secretory processes. Also, rates of Cl− and I− absorption in DRA‐knockout mice were decreased by 79% and 50%, respectively, in the ceca, and 65% and 34% in the distal colon, suggesting this Cl− transporter may have a previously unrecognized role in I− uptake. In summary, we demonstrated 125I− is an adequate, cost‐effective alternative for tracing unidirectional Clfluxes across the intestinal epithelium. For example, the amount of 36Cl− used in this study cost $536 compared to a mere $4 worth of 125I−, thus making future research into epithelial Cltransport accessible. In addition, this study also provides insight into mechanisms of net Clsecretion and suggests that DRA may have a role in intestinal I− absorption.Support or Funding InformationThis study was supported by grants DK088892 and DK108755 to M. Hatch from the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.

The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca2+ concentration. The protein is broadly expressed and contributes to diverse physiological processes, including transepithelial chloride transport and the control of electrical signalling in smooth muscles and certain neurons. As a member of the TMEM16 (or anoctamin) family of membrane proteins, TMEM16A is closely related to paralogues that function as scramblases, which facilitate the bidirectional movement of lipids across membranes. The unusual functional diversity of the TMEM16 family and the relationship between two seemingly incompatible transport mechanisms has been the focus of recent investigations. Previous breakthroughs were obtained from the X-ray structure of the lipid scramblase of the fungus Nectria haematococca (nhTMEM16), and from the cryo-electron microscopy structure of mouse TMEM16A at 6.6 Å (ref. 14). Although the latter structure disclosed the architectural differences that distinguish ion channels from lipid scramblases, its low resolution did not permit a detailed molecular description of the protein or provide any insight into its activation by Ca2+. Here we describe the structures of mouse TMEM16A at high resolution in the presence and absence of Ca2+. These structures reveal the differences between ligand-bound and ligand-free states of a calcium-activated chloride channel, and when combined with functional experiments suggest a mechanism for gating. During activation, the binding of Ca2+ to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening. Our study describes a process that is unique among channel proteins, but one that is presumably general for both functional branches of the TMEM16 family.

Molecular Mechanism of TMEM16A CA2+-Activated Chloride Channel Desensitization

TMEM16A forms the long-sought after calcium-activated chloride channel (CaCC) that is highly expressed in various tissues such as dorsal root ganglia (DRGs), epithelial and smooth muscle cells. Extensive studies have demonstrated that the TMEM16A-CaCC plays important roles in mediating nociception, trans-epithelial chloride transport and smooth muscle contraction. Whilst the activation mechanism of the TMEM16A-CaCC has been extensively explored, how the channel activity is regulated in different cell types remains elusive. By studying the molecular mechanism of TMEM16A-CaCC desensitization, a well-documented characteristic of CaCCs, we aim to understand how the TMEM16A-CaCC is regulated at molecular and cellular levels. Our findings will not only illustrate the desensitization mechanism of the TMEM16A-CaCC channel, but add new insights on understanding the channel activation mechanism. As the desensitization severely hampers the biophysical characterization of TMEM16A-CaCC properties, our findings will provide a solution to prevent CaCC desensitization and thus facilitate the structure-function study of the TMEM16 channels.

A Specific Haplotype Framework Surrounds the Omani Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) Mutation S549R

Cystic Fibrosis (CF) is an autosomal recessive disorder affecting the chloride transport in mucus-producing epithelial cells. The disease is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), which is responsible for trans-epithelial chloride transport. Approximately 1900 mutations and gene variants of the CFTR have been described. The spectrum of major White-European mutations includes F508del, G542X, G551D and N1303K. F508del is the most common CF-causing mutation, found in approximately 70% of all CF patients worldwide. The spectrum of CF mutations of Arab populations is under-investigated. However, initial molecular-epidemiological studies indicate the existence of specific CF mutation clusters within geographical regions in the Middle East, suggesting specific distributions of CF mutation carrying chromosomes in this part of the world. We showed that the world-wide rare CF mutation S549R is the predominant disease causing mutation in the Omani population. We reported that S549R, together with two other identified mutations, F508del and the rare private mutation V392G, are genetically linked to the exonic methionine polymorphism c.1408A>G; p.Met470Val at exon 10 and the intronic dimorphic 4-bp GATT 6-repeat at intron 6, c.744_33GATT[6_8]. We detected three haplotypes in 28 alleles of the Omani CF cohort and 408 alleles of our control cohort of unrelated and unaffected Omani volunteers. The CF disease associated haplotype consisting of an M allele and a 6-repeat expansion, occurred with an allele frequency of only 0.174 in the normal Omani population. The discriminative power of the haplotype was attributed to the intronic dimorphic 4-bp GATT 6-repeat. Furthermore, we found only one mutation, c.1733_1734delTA in the Omani CF cohort which deviated from the rule and shared the most common haplotype, a V allele and a 7-repeat extension, with the normal population.

Sinupret activates CFTR and TMEM16A-dependent transepithelial chloride transport and improves indicators of mucociliary clearance.

IntroductionWe have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl−) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.MethodsPrimary murine nasal septal epithelial (MNSE) [wild type (WT) and transgenic CFTR−/−], human sinonasal epithelial (HSNE), WT CFTR-expressing CFBE and TMEM16A-expressing HEK cultures were utilized for the present experiments. CBF and ASL depth measurements were performed. Mechanisms underlying transepithelial Cl− transport were determined using pharmacologic manipulation in Ussing chambers, Fura-2 intracellular calcium [Ca2+]i imaging, cAMP signaling, regulatory domain (R-D) phosphorylation of CFTR, and excised inside out and whole cell patch clamp analysis.ResultsSinupret-mediated Cl− secretion [ΔISC(µA/cm2)] was pronounced in WT MNSE (20.7+/−0.9 vs. 5.6+/−0.9(control), p<0.05), CFTR−/− MNSE (10.1+/−1.0 vs. 0.9+/−0.3(control), p<0.05) and HSNE (20.7+/−0.3 vs. 6.4+/−0.9(control), p<0.05). The formulation activated Ca2+ signaling and TMEM16A channels, but also increased CFTR channel open probability (Po) without stimulating PKA-dependent pathways responsible for phosphorylation of the CFTR R-domain and resultant Cl− secretion. Sinupret also enhanced CBF and ASL depth.ConclusionSinupret stimulates CBF, promotes transepithelial Cl− secretion, and increases ASL depth in a manner likely to enhance MCC. Our findings suggest that direct stimulation of CFTR, together with activation of Ca2+-dependent TMEM16A secretion account for the majority of anion transport attributable to Sinupret. These studies provide further rationale for using robust Cl− secretagogue based therapies as an emerging treatment modality for common respiratory diseases of MCC including acute and chronic bronchitis and CRS.

Treatment with 17-allylamino-17-demethoxygeldanamycin ameliorated symptoms of Bartter syndrome type IV caused by mutated Bsnd in mice

Mutations of BSND, which encodes barttin, cause Bartter syndrome type IV. This disease is characterized by salt and fluid loss, hypokalemia, metabolic alkalosis, and sensorineural hearing impairment. Barttin is the β-subunit of the ClC-K chloride channel, which recruits it to the plasma membranes, and the ClC-K/barttin complex contributes to transepithelial chloride transport in the kidney and inner ear. The retention of mutant forms of barttin in the endoplasmic reticulum (ER) is etiologically linked to Bartter syndrome type IV. Here, we report that treatment with 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, enhanced the plasma membrane expression of mutant barttins (R8L and G47R) in Madin–Darby canine kidney cells. Administration of 17-AAG to BsndR8L/R8L knock-in mice elevated the plasma membrane expression of R8L in the kidney and inner ear, thereby mitigating hypokalemia, metabolic alkalosis, and hearing loss. These results suggest that drugs that rescue ER-retained mutant barttin may be useful for treating patients with Bartter syndrome type IV.

Normalization of Sweat Chloride Concentration and Clinical Improvement With Ivacaftor in a Patient With Cystic Fibrosis With Mutation S549N

The cystic fibrosis (CF) protein forms an anion channel in epithelial cells, and the absence or defective function of this channel results in the clinical manifestations of CF. CF is an autosomal recessive disorder, and its many disease-causing mutations divide into five or six classes. There are 10 known class 3 gating mutations, the most common of which is G551D. Ivacaftor is a drug that in vitro increases open time and transepithelial chloride transport in all 10 gating mutations, but it is approved for use only in patients with the G551D mutation. We report complete normalization of sweat chloride concentration and rapid clinical improvement over 6 weeks of treatment with ivacaftor in a patient with CF with the gating mutation S549N. The findings suggest that ivacaftor should be considered for use in patients with any of the known gating mutations.

Development, clinical utility, and place of ivacaftor in the treatment of cystic fibrosis

Cystic fibrosis (CF) is a life-limiting, multisystem disease characterized by thick viscous secretions leading to recurrent lung infections, bronchiectasis, and progressive deterioration in lung function. CF is caused by loss or dysfunction of the CF transmembrane conductance regulator (CFTR) protein which is responsible for transepithelial chloride and water transport. Improved understanding of CFTR protein dysfunction has allowed the development of mutation-specific small-molecule compounds which directly target the underlying CFTR defect. Ivacaftor is the first licensed small-molecule compound for CF patients which targets the CFTR gating mutation Gly551Asp (previously termed G551D) and has the potential to be truly disease-modifying. Ivacaftor is an oral medication given twice daily and has shown benefit in terms of an increase in lung function, decreased sweat chloride, weight gain, improvement in patient-reported quality of life, and reduction in number of respiratory exacerbations in clinical trials. Although ivacaftor is currently only licensed for use in approximately 5% of the CF population (those who have at least one Gly551Asp mutation), the developmental pathway established by ivacaftor paves the way for other CFTR modulators that may benefit many more patients. In particular, a CFTR modulator for those with the Phe508del deletion (previously ∆F508) would allow 90% of the CF population to benefit from disease-modifying treatment.

The Novel Dry Extract BNO 1011 Stimulates Chloride Transport and Ciliary Beat Frequency in Human Respiratory Epithelial Cultures

Herbal remedies predate written history and continue to be used more frequently than conventional pharmaceutical medications. The novel dry extract BNO 1011 is based on a combination of five herbs that is used to treat acute and chronic rhinosinusitis. We evaluated the pharmacologic effects of the novel dry extract BNO 1011 on human respiratory epithelial cultures specifically addressing electrolyte transport and cilia beat frequency (CBF). Well-differentiated human bronchial epithelial cultures grown at an air-liquid interface were treated on the apical or basolateral surface with varying concentrations of dry extract BNO 1011. Changes in transepithelial sodium and chloride transport were determined in Ussing chambers under voltage-clamped conditions. Changes in CBF were determined using the Sissons-Ammons Video Analysis system (Ammons Engineering, Mt. Morris, MI). When applied to the apical surface, dry extract BNO 1011 activated forskolin-stimulated chloride secretion and ciliary beat in a dose-dependent fashion. Basolateral application of dry extract BNO 1011 did not alter the measured physiological properties. Apical application of dry extract BNO 1011 stimulates both chloride secretion and CBF and therefore may augment mucociliary clearance.

A Synthetic Chloride Channel Restores Chloride Conductance in Human Cystic Fibrosis Epithelial Cells

Mutations in the gene-encoding cystic fibrosis transmembrane conductance regulator (CFTR) cause defective transepithelial transport of chloride (Cl−) ions and fluid, thereby becoming responsible for the onset of cystic fibrosis (CF). One strategy to reduce the pathophysiology associated with CF is to increase Cl− transport through alternative pathways. In this paper, we demonstrate that a small synthetic molecule which forms Cl− channels to mediate Cl− transport across lipid bilayer membranes is capable of restoring Cl− permeability in human CF epithelial cells; as a result, it has the potential to become a lead compound for the treatment of human diseases associated with Cl− channel dysfunction.

Immunomodulatory activity of vardenafil on induced lung inflammation in cystic fibrosis mice

BackgroundWe tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. MethodsInflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14mg/kg) was evaluated. ResultsA latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1β was observed in the CF group only. ConclusionVardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.

Gastrin inhibits a novel, pathological colon cancer signaling pathway involving EGR1, AE2, and P-ERK

Human anion exchanger 2 (AE2) is a plasma membrane protein that regulates intracellular pH and cell volume. AE2 contributes to transepithelial transport of chloride and bicarbonate in normal colon and other epithelial tissues. We now report that AE2 overexpression in colon cancer cells is correlated with expression of the nuclear proliferation marker, Ki67. Survival analysis of 24 patients with colon cancer in early stage or 33 patients with tubular adenocarcinoma demonstrated that expression of AE2 is correlated with poor prognosis. Cellular and molecular experiments indicated that AE2 expression promoted proliferation of colon cancer cells. In addition, we found that transcription factor EGR1 underlies AE2 upregulation and the AE2 sequester p16INK4a (P16) in the cytoplasm of colon cancer cells. Cytoplasmic P16 enhanced ERK phosphorylation and promoted proliferation of colon cancer cells. Gastrin inhibited proliferation of colon cancer cells by suppressing expression of EGR1 and AE2 and by blocking ERK phosphorylation. Taken together, our data describe a novel EGR1/AE2/P16/P-ERK signaling pathway in colon carcinogenesis, with implications for pathologic prognosis and for novel therapeutic approaches.

Implications for health and disease in the genetic signature of the Ashkenazi Jewish population.

BackgroundRelatively small, reproductively isolated populations with reduced genetic diversity may have advantages for genomewide association mapping in disease genetics. The Ashkenazi Jewish population represents a unique population for study based on its recent (< 1,000 year) history of a limited number of founders, population bottlenecks and tradition of marriage within the community. We genotyped more than 1,300 Ashkenazi Jewish healthy volunteers from the Hebrew University Genetic Resource with the Illumina HumanOmni1-Quad platform. Comparison of the genotyping data with that of neighboring European and Asian populations enabled the Ashkenazi Jewish-specific component of the variance to be characterized with respect to disease-relevant alleles and pathways.ResultsUsing clustering, principal components, and pairwise genetic distance as converging approaches, we identified an Ashkenazi Jewish-specific genetic signature that differentiated these subjects from both European and Middle Eastern samples. Most notably, gene ontology analysis of the Ashkenazi Jewish genetic signature revealed an enrichment of genes functioning in transepithelial chloride transport, such as CFTR, and in equilibrioception, potentially shedding light on cystic fibrosis, Usher syndrome and other diseases over-represented in the Ashkenazi Jewish population. Results also impact risk profiles for autoimmune and metabolic disorders in this population. Finally, residual intra-Ashkenazi population structure was minimal, primarily determined by class 1 MHC alleles, and not related to host country of origin.ConclusionsThe Ashkenazi Jewish population is of potential utility in disease-mapping studies due to its relative homogeneity and distinct genomic signature. Results suggest that Ashkenazi-associated disease genes may be components of population-specific genomic differences in key functional pathways.

Generation and analyses of R8L barttin knockin mouse

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

CFTR-deficiency renders mice highly susceptible to cutaneous symptoms during mite infestation

Pruritus, also known as itch, is a sensation that causes a desire to scratch. Prolonged scratching exacerbates skin lesions in several skin diseases such as atopic dermatitis. Here, we identify the cystic fibrosis transmembrane conductance regulator (CFTR/Cftr), an integral membrane protein that mediates transepithelial chloride transport, as a determinant factor in mice for the susceptibility to several cutaneous symptoms during mite infestation. Mice that endogenously express dysfunctional Cftr (CftrΔF508/ΔF508) show significant increase of scratching behavior and skin fibrosis after mite exposure. These phenotypes were due to the increased expression of nerve growth factor (NGF) that augments the sensitization of peripheral nerve fibers. Moreover, protein gene product 9.5 (PGP9.5)-positive neurites were abundant in the epidermis of mite-infested CftrΔF508/ΔF508 mice. Furthermore, mite-infested Cftr+/+ mice orally administered with a chloride channel inhibitor glibenclamide had higher scratching count and increased level of NGF than vehicle-treated mice. Consistently, mite extract-exposed primary and transformed human keratinocytes, treated with CFTR inhibitor, had significantly higher level of NGF mRNA compared with vehicle-treated, mite extract-exposed cells. These results reveal that CFTR in keratinocytes plays a critical role for the regulation of peripheral nerve function and pruritus sensation, and suggest that CftrΔF508/ΔF508 mice may serve as a novel mouse model that represents NGF-dependent generation of pruritus.

The bioflavonoid compound, sinupret, stimulates transepithelial chloride transport in vitro and in vivo

Dehydration of airway surface liquid (ASL) disrupts normal mucociliary clearance in sinonasal epithelium leading to chronic rhinosinusitis. Abnormal chloride (Cl(-)) transport is one mechanism that contributes to this disorder, as demonstrated by the disease cystic fibrosis. Identifying safe compounds that stimulate transepithelial Cl(-) transport is critical to improving hydration of the ASL and promoting mucociliary transport. Sinupret (Bionorica, LLC, San Clemente, CA), a combination of naturally occurring bioflavonoids, is a widely used treatment for respiratory ailments in Europe. However, the effects of Sinupret on target respiratory epithelium have yet to be fully investigated. The present study evaluated the mechanisms underlying this bioflavonoid therapeutic on transepithelial Cl(-) transport in respiratory epithelium. In vitro and in vivo investigation. Well characterized murine nasal septal epithelial (MNSE) cultures, and murine nasal potential difference (NPD) techniques were used to evaluate the effects of Sinupret on Cl(-) secretion. The change in Sinupret-stimulated current (Delta I(SC) expressed as microA/cm(2)) in MNSE, representing Cl(-) secretion, was significantly increased when compared to controls (19.04 + or - 1.67 microA/cm(2) vs. 1.8 + or - 0.35 microA/cm(2), respectively; P = .00005). Transepithelial Cl(-) transport measured in the murine NPD in vivo assay (n = 42) was also significantly enhanced when compared to controls (-0.8 mV vs. -0.9 mV; P = .0004). Importantly, Sinupret-stimulated Cl(-) transport was substantially more robust in vivo than forskolin, a compound among the strongest known cystic fibrosis transmembrane conductance regulator activators (-3.8 mV vs. -1.65 mV; P = .01). Sinupret strongly activates transepithelial Cl(-) secretion through a mechanism known to hydrate the ASL of respiratory epithelium. This is one means by which the medication is likely to exert therapeutic benefit.