Pulmonary vasoconstrictor action of KCNQ potassium channel blockers

KCNQ channels have been widely studied in the nervous system, heart and inner ear, where they have important physiological functions. Recent reports indicate that KCNQ channels may also be expressed in portal vein where they are suggested to influence spontaneous contractile activity. The biophysical properties of K+ currents mediated by KCNQ channels resemble a current underlying the resting K+ conductance and resting potential of pulmonary artery smooth muscle cells. We therefore investigated a possible role of KCNQ channels in regulating the function of pulmonary arteries by determining the ability of the selective KCNQ channel blockers, linopirdine and XE991, to promote pulmonary vasoconstriction. Linopirdine and XE991 both contracted rat and mouse pulmonary arteries but had little effect on mesenteric arteries. In each case the maximum contraction was almost as large as the response to 50 mM K+. Linopirdine had an EC50 of around 1 μM and XE991 was almost 10-fold more potent. Neither removal of the endothelium nor exposure to phentolamine or α,β-methylene ATP, to block α1-adrenoceptors or P2X receptors, respectively, affected the contraction. Contraction was abolished in Ca2+-free solution and in the presence of 1 μM nifedipine or 10 μM levcromakalim

A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane

BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix. METHODOLOGY/PRINCIPAL FINDINGS: Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure

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

The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1 : contributions of the pore domains

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb+ or Na+ replaced extracellular K+. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels nonselective. We interpret the effects of these—and of other mutations—in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue

Lubiprostone Stimulates Duodenal Bicarbonate Secretion in Rats

Lubiprostone, a bicyclic fatty acid, is used for the treatment of chronic constipation. No published study has addressed the effect of lubiprostone on intestinal ion secretion in vivo. The aim of this study was to test the hypothesis that lubiprostone augments duodenal HCO3 − secretion (DBS). Rat proximal duodenal loops were perfused with pH 7.0 Krebs, control vehicle (medium-chain triglycerides), or lubiprostone (0.1–10 μM). We measured DBS with flow-through pH and CO2 electrodes, perfusate [Cl−] with a Cl− electrode, and water flux using a non-absorbable ferrocyanide marker. Some rats were pretreated with a potent, selective CFTR antagonist, CFTRinh-172 (1 mg/kg, ip), 1 h before experiments. Perfusion of lubiprostone concentration dependently increased DBS, whereas net Cl− output and net water output were only increased at 0.1 μM, compared with vehicle. CFTRinh-172 reduced lubiprostone (10 μM)-induced DBS increase, whereas net Cl− output was also unchanged. Nevertheless, CFTRinh-172 reduced basal net water output, which was reversed by lubiprostone. Furthermore, lubiprostone-induced DBS was inhibited by EP4 receptor antagonist, not by an EP1/2 receptor antagonist or by indomethacin pretreatment. In this first study of the effect of lubiprostone on intestinal ion secretion in vivo, lubiprostone stimulated CFTR-dependent DBS without changing net Cl− secretion. This effect supports the hypothesis that Cl− secreted by CFTR is recycled across the apical membrane by anion exchangers. Recovery of water output during CFTR inhibition suggests that lubiprostone may improve the intestinal phenotype in CF patients. Furthermore, increased DBS suggests that lubiprostone may protect the duodenum from acid-induced injury via EP4 receptor activation

Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes

Abnormal spatial diffusion of Ca2+ in F508del-CFTR airway epithelial cells

<p>Abstract</p> <p>Background</p> <p>In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG) and induces Ca²⁺release from endoplasmic reticulum (ER) stores.</p> <p>Methods</p> <p>In the present study, we monitored the cytosolic Ca²⁺transients using the UV light photolysis technique to uncage caged Ca²⁺or caged IP₃into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca²⁺receptors present in the ER, and measured their Ca²⁺dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin).</p> <p>Results</p> <p>We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP₃R) dependent-Ca²⁺response following both correcting treatments compared to uncorrected cells in such a way that Ca²⁺responses (CF+treatment <it>vs </it>wild-type cells) were normalized. This normalization of the Ca²⁺rate does not affect the activity of Ca²⁺-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP₃R1, we observed a decrease of the implication of IP₃R1 in the Ca²⁺response in CF corrected cells. We observed a similar Ca²⁺mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP₃R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell.</p> <p>Conclusion</p> <p>These results suggest reversal of the IP₃R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP₃R Ca²⁺release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.</p

Ileal mucosal bile acid absorption is increased in Cftr knockout mice

BACKGROUND: Excessive loss of bile acids in stool has been reported in patients with cystic fibrosis. Some data suggest that a defect in mucosal bile acid transport may be the mechanism of bile acid malabsorption in these individuals. However, the molecular basis of this defect is unknown. This study examines the expression of the ileal bile acid transporter protein (IBAT) and rates of diffusional (sodium independent) and active (sodium dependent) uptake of the radiolabeled bile acid taurocholate in mice with targeted disruption of the cftr gene. METHODS: Wild-type, heterozygous cftr (+/-) and homozygous cftr (-/-) mice were studied. Five one-cm segments of terminal ileum were excised, everted and mounted onto thin stainless steel rods and incubated in buffer containing tracer (3)H-taurocholate. Simultaneously, adjacent segments of terminal ileum were taken and processed for immunohistochemistry and Western blots using an antibody against the IBAT protein. RESULTS: In all ileal segments, taurocholate uptake rates were fourfold higher in cftr (-/-) and two-fold higher in cftr (+/-) mice compared to wild-type mice. Passive uptake was not significantly higher in cftr (-/-) mice than in controls. IBAT protein was comparably increased. Immuno-staining revealed that the greatest increases occurred in the crypts of cftr (-/-) animals. CONCLUSIONS: In the ileum, IBAT protein densities and taurocholate uptake rates are elevated in cftr (-/-) mice > cftr (+/-) > wild-type mice. These findings indicate that bile acid malabsorption in cystic fibrosis is not caused by a decrease in IBAT activity at the brush border. Alternative mechanisms are proposed, such as impaired bile acid uptake caused by the thick mucus barrier in the distal small bowel, coupled with a direct negative regulatory role for cftr in IBAT function