Small-conductance Cl? channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

1992 ◽  
Vol 421 (2-3) ◽  
pp. 238-246 ◽  
Author(s):  
K. Kunzelmann ◽  
R. Kubitz ◽  
M. Grolik ◽  
R. Warth ◽  
R. Greger
Keyword(s):  
Cell Swelling ◽  
Ht29 Cells ◽  
Cl Channels ◽  
Hypotonic Cell Swelling ◽  
Small Conductance

Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling

1994 ◽  
Vol 428 (1) ◽  
pp. 76-83 ◽  
Author(s):  
K. Kunzelmann ◽  
N. Allert ◽  
R. Kubitz ◽  
W. V. Breuer ◽  
Z. I. Cabantchik ◽  
...  
Keyword(s):  
Chloride Conductance ◽  
Cell Swelling ◽  
Ht29 Cells ◽  
Hypotonic Cell Swelling

Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule

1994 ◽  
Vol 266 (4) ◽  
pp. F543-F553 ◽  
Author(s):  
V. Poncet ◽  
M. Tauc ◽  
M. Bidet ◽  
P. Poujeol
Keyword(s):  
Chloride Channels ◽  
Apical Membrane ◽  
Primary Cultures ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Current Voltage ◽  
Cl Channels ◽  
Inside Out ◽  
Small Conductance

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.

scholarly journals Volume-regulated Cl− current: contributions of distinct Cl− channels and localized Ca2+ signals

2019 ◽  
Vol 317 (3) ◽  
pp. C466-C480 ◽  
Author(s):  
Yani Liu ◽  
Huiran Zhang ◽  
Hongchao Men ◽  
Yuwei Du ◽  
Ziqian Xiao ◽  
...  
Keyword(s):  
Anion Channel ◽  
Fluorescent Imaging ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Anoctamin 1 ◽  
Component Cell ◽  
Intracellular Ca ◽  
Cl Channels ◽  
A Cell ◽  
Necessary And Sufficient

The swelling-activated chloride current ( ICl,swell) is induced when a cell swells and plays a central role in maintaining cell volume in response to osmotic stress. The major contributor of ICl,swell is the volume-regulated anion channel (VRAC). Leucine-rich repeat containing 8A (LRRC8A; SWELL1) was recently identified as an essential component of VRAC, but the mechanisms of VRAC activation are still largely unknown; moreover, other Cl− channels, such as anoctamin 1 (ANO1), were also suggested to contribute to ICl,swell. In this present study, we investigated the roles of LRRC8A and ANO1 in activation of ICl,swell; we also explored the role of intracellular Ca2+ in ICl,swell activation. We used a CRISPR/Cas9 gene editing approach, electrophysiology, live fluorescent imaging, selective pharmacology, and other approaches to show that both LRRC8A and ANO1 can be activated by cell swelling in HEK293 cells. Yet, both channels contribute biophysically and pharmacologically distinct components to ICl,swell, with LRRC8A being the major component. Cell swelling induced oscillatory Ca2+ transients, and these Ca2+ signals were required to activate both the LRRC8A- and ANO1-dependent components of ICl,swell. Both ICl,swell components required localized rather than global Ca2+ for activation. Interestingly, while intracellular Ca2+ was necessary and sufficient to activate ANO1, it was necessary but not sufficient to activate LRRC8A-mediated currents. Finally, Ca2+ transients linked to the ICl,swell activation were mediated by the G protein-coupled receptor-independent PLC isoforms.

Cell volume analysis of gramicidin-treated eccrine clear cells to study regulation of Cl channels

1993 ◽  
Vol 265 (4) ◽  
pp. C1090-C1099 ◽  
Author(s):  
M. Ohtsuyama ◽  
Y. Suzuki ◽  
G. Samman ◽  
F. Sato ◽  
K. Sato
Keyword(s):  
Cell Volume ◽  
Membrane Conductance ◽  
Cell Swelling ◽  
Tetraacetic Acid ◽  
Sensitive Assay ◽  
Current Clamp ◽  
Volume Analysis ◽  
Clear Cells ◽  
Cyclic Monophosphate ◽  
Cl Channels

Using voltage-current-clamp methods, we determined membrane potentials, relative ionic permeability, and membrane conductance of gramicidin (GC)-treated freshly dissociated eccrine clear cells. GC depolarized the membrane potential by 58 mV, increased the membrane conductance progressively over the time of exposure (mean of 1.7 times at 60 s and 4.6 times at 3 min), and increased the Na conductance of the membrane (from near 0 in control to 0.75 nS after GC). Image analysis coupled with GC treatment was then employed to study the regulation of Cl channels based on the premise that cell swelling was due to activation of Cl channels. Cell swelling was stimulated by methacholine (MCh, 3 microM) in the presence of GC. GC+MCh-induced cell swelling was inhibited by atropine, low extracellular Ca ([Ca]o < 1 nM), or removal of Cl. Thus MCh-induced cell swelling is most likely due to Ca-dependent activation of Cl channels. Isoproterenol (Iso), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, and forskolin also caused cell swelling in the presence of GC. Iso-induced cell swelling was abolished in a Cl-free medium and by diphenylamine-2-carboxylic acid, indicating that it is caused by adenosine 3',5'-cyclic monophosphate (cAMP)-mediated activation of Cl channels. Cl channels stimulated by MCh, but not those stimulated by Iso, were inhibited by preexposure to a low-Ca medium [nominally Ca free + 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, [Ca]o < 1 nM] for 20 s, suggesting that Ca-stimulated Cl channels are distinct from cAMP-dependent Cl channels. cAMP-stimulated Cl channels were, however, inhibited when the cells were exposed to the low-Ca medium for 60 s. The simple cell volume analysis of GC-treated cells is a sensitive assay system for both Ca- and cAMP-dependent Cl channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels

1996 ◽  
Vol 270 (6) ◽  
pp. C1790-C1798 ◽  
Author(s):  
Y. Chen ◽  
S. M. Simasko ◽  
J. Niggel ◽  
W. J. Sigurdson ◽  
F. Sachs
Keyword(s):  
Volume Regulation ◽  
Cell Swelling ◽  
Gh3 Cells ◽  
Membrane Tension ◽  
Hypotonic Cell Swelling ◽  
Stretch Activated Channels ◽  
Ca2 Channels ◽  
Hypotonic Swelling ◽  
Volume Decrease

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

scholarly journals Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

1999 ◽  
Vol 276 (2) ◽  
pp. C328-C336 ◽  
Author(s):  
Christopher M. Gillen ◽  
Bliss Forbush
Keyword(s):  
Cell Swelling ◽  
Primary Role ◽  
Functional Interaction ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Hek Cells ◽  
293 Cells ◽  
Hypotonic Cell Swelling ◽  
The Relationship ◽  
Fold Activation

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

scholarly journals A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers.

1990 ◽  
Vol 96 (4) ◽  
pp. 707-733 ◽  
Author(s):  
G L Lukács ◽  
E Moczydlowski
Keyword(s):  
Single Channel ◽  
Disulfonic Acid ◽  
Ph Change ◽  
Planar Bilayers ◽  
Voltage Range ◽  
Cl Channel ◽  
Voltage Dependent ◽  
Cl Channels ◽  
Transport Inhibitors ◽  
Small Conductance

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.

scholarly journals Cholesterol Depletion Facilitates Recovery from Hypotonic Cell Swelling in CHO Cells

2011 ◽  
Vol 28 (6) ◽  
pp. 1247-1254 ◽  
Author(s):  
Gregory B. Kowalsky ◽  
Derek Beam ◽  
Myung J. Oh ◽  
Frederick Sachs ◽  
Susan Z. Hua ◽  
...  
Keyword(s):  
Cho Cells ◽  
Cell Swelling ◽  
Cholesterol Depletion ◽  
Hypotonic Cell Swelling
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