405: Calcium activated chloride channel regulator 1 VWA domain can potentiate TMEM16A anion channel in primary CF airway cells and tissues to enhance airway defense properties

Ion channels are fundamental to gastrointestinal pacemaking by interstitial cells of Cajal (ICC). Previously, we have recorded a high-conductance chloride channel (HCCC) from ICC, both in culture and in situ, associated with the myenteric plexus. The biophysical properties of the HCCC (conductance, subconductances, voltage- and time-dependent inactivation) suggest it is a member of a class called the maxi-anion channels. In this study we further investigated the properties of the HCCC in situ. Our main finding was that the HCCC is not strictly a chloride channel but has a relative sodium-chloride permeability (PNa/Cl) of 0.76 to 1.64 (depending on the method of measurement). Therefore, we have renamed the HCCC the “maxi-channel.” A maxi-channel was also expressed by pericytes associated with the vasculature near the myenteric plexus. This had a lower PNa/Cl (0.33 to 0.49, depending on the method of measurement) but similar conductance (326 ± 7 vs. 316 ± 24 pS for ICC). This is the first report of cation permeability equaling anion permeability in a maxi-anion channel. As such, the properties of the maxi-channels described in this article may have implications for the maxi-anion channel field, as well as for studies of their role in ICC and pericytes.

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Superoxide Flux in Endothelial Cells via the Chloride Channel-3 Mediates Intracellular Signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0830 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2002-2012 ◽

Cited By ~ 120

Author(s):

Brian J. Hawkins ◽

Muniswamy Madesh ◽

C. J. Kirkpatrick ◽

Aron B. Fisher

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Chloride Channel ◽

Intracellular Signaling ◽

Anion Channel ◽

Transmembrane Flux ◽

Cell Plasma ◽

Intracellular Ca ◽

Cellular Apoptosis ◽

Extracellular Side

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O2.−) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O2.−, pulmonary microvascular endothelial cells were preloaded with the O2.−-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O2.−resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O2.−triggered Ca2+release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O2.−production and cellular apoptosis. These “signaling” effects of O2.−were prevented by DIDS treatment, by depletion of intracellular Ca2+stores with thapsigargin and by chelation of intracellular Ca2+. This study demonstrates that O2.−flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca2+release, which activates mitochondrial O2.−generation.

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Evidence for a Superoxide Permeability Pathway in Endosomal Membranes

Molecular and Cellular Biology ◽

10.1128/mcb.02038-07 ◽

2008 ◽

Vol 28 (11) ◽

pp. 3700-3712 ◽

Cited By ~ 66

Author(s):

Davis R. Mumbengegwi ◽

Qiang Li ◽

Canhui Li ◽

Christine E. Bear ◽

John F. Engelhardt

Keyword(s):

Chloride Channel ◽

Anion Channel ◽

Disulfonic Acid ◽

Channel Blockers ◽

Luminescent Probes ◽

Redox Activation ◽

Early Endosomes ◽

Signaling Events ◽

Endosomal Membranes ◽

Immunoaffinity Isolation

ABSTRACT The compartmentalized production of superoxide (·O2 −) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-κB following interleukin 1β (IL-1β) stimulation. It remains unclear how ·O2 − produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated ·O2 − movement out of IL-1β-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-κB activation. The relative outward permeability of NADPH-dependent ·O2 − from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for ·O2 −. In these studies, ∼60% of ·O2 − efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that ·O2 − and Cl− are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1β-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-κB following IL-1β stimulation. Together, these results suggest that ·O2 − exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of ·O2 − at the endosomal surface may produce the localized H2O2 required for redox-activation of NF-κB.

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The activity of plant inner membrane anion channel (PIMAC) can be performed by a chloride channel (CLC) protein in mitochondria from seedlings of maize populations divergently selected for cold tolerance

Journal of Bioenergetics and Biomembranes ◽

10.1007/s10863-011-9386-z ◽

2011 ◽

Vol 43 (6) ◽

pp. 611-621 ◽

Cited By ~ 14

Author(s):

Elisabetta Tampieri ◽

Elena Baraldi ◽

Francesco Carnevali ◽

Elisabetta Frascaroli ◽

Aurelio De Santis

Keyword(s):

Cold Tolerance ◽

Chloride Channel ◽

Anion Channel ◽

Inner Membrane ◽

Maize Populations ◽

Inner Membrane Anion Channel

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Development of a Colorimetric Method for Functional Chloride Channel Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104266740 ◽

2004 ◽

Vol 9 (7) ◽

pp. 607-613 ◽

Cited By ~ 6

Author(s):

Weimin Tang ◽

Mary Jo Wildey

Keyword(s):

Chloride Channel ◽

High Throughput Screening ◽

Colorimetric Detection ◽

Colorimetric Method ◽

Anion Channel ◽

Aminobutyric Acid ◽

Anion Channels ◽

Acid Type ◽

A Cell ◽

Inwardly Rectifying

Anion channels play significant physiological roles in humans and animals. However, the effort of screening for anion channel modulators was limited by the available assay technologies. This report discusses the development of a cell-based functional chloride channel assay using iodine as the chloride channel functional indicator. Iodine concentrations were measured with modified Sandell-Kolthoff reaction using colorimetric detection. The assay was rapid and quantitative. When WSS-1 cells were activated by γ-aminobutyric acid (GABA) in the condition that γ-aminobutyric acid type A receptor (GABAA receptor) conducted outwardly rectifying chloride channel function, the EC50 of GABA was 7.69 μM. IC50 swere 0.53 μM for bicuculline and 3.1 μM for picrotoxin, respectively, in the presence of 10 μM GABA. When Capan-1 cells were activated by forskolin, the EC50 was 0.14 μM. The assay can also be applied to inwardly rectifying anion channels as exemplified by GABAA channel with an EC50 of 294 μM. Thus, the assay is universal and reliable and can be used for anion channel high-throughput screening.

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TMC4 is a novel chloride channel involved in high-concentration salt taste sensation

The Journal of Physiological Sciences ◽

10.1186/s12576-021-00807-z ◽

2021 ◽

Vol 71 (1) ◽

Cited By ~ 2

Author(s):

Yoichi Kasahara ◽

Masataka Narukawa ◽

Yoshiro Ishimaru ◽

Shinji Kanda ◽

Chie Umatani ◽

...

Keyword(s):

Chloride Channel ◽

Anion Channel ◽

Chloride Ions ◽

Glossopharyngeal Nerve ◽

Molecular Entity ◽

Taste Sensation ◽

High Concentration ◽

Transmembrane Channel ◽

Voltage Dependent ◽

Anion Channel Blocker

Abstract“Salty taste” sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl− receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl− channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl−. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

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