scholarly journals Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells

Membranes ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 723
Author(s):  
Khaoula Talbi ◽  
Jiraporn Ousingsawat ◽  
Raquel Centeio ◽  
Rainer Schreiber ◽  
Karl Kunzelmann
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Hek293 Cells ◽  
K Channel ◽  
Airway Epithelial ◽  
Colonic Epithelial Cells ◽  
Cl Channel ◽  
Endogenous Expression ◽  
Regulatory Properties ◽  
Limit Evidence

Regulation of the Ca2+-activated Cl− channel TMEM16A by Ca2+/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca2+/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca2+/CAM-regulated K+ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca2+ sensitivity of the Cl− channel. Enhanced Ca2+ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca2+ levels. The present results correspond well to a recent report that demonstrates a Ca2+-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca2+-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.

Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C243-C251 ◽  
Author(s):  
M. E. Egan ◽  
E. M. Schwiebert ◽  
W. B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Incubation Temperature ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Cl Channel ◽  
Channel Expression

When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF)-affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells.

Basolateral K+ channels in airway epithelia. II. Role in Cl- secretion and evidence for two types of K+ channel

1990 ◽  
Vol 258 (6) ◽  
pp. L343-L348 ◽  
Author(s):  
J. D. McCann ◽  
M. J. Welsh
Keyword(s):  
Epithelial Cells ◽  
Time Course ◽  
Basolateral Membrane ◽  
Airway Epithelial Cells ◽  
K Channel ◽  
Airway Epithelial ◽  
K Channels ◽  
Cl Secretion ◽  
Transient Component ◽  
Permeable Supports

We previously described a Ca2(+)-activated K+ channel (KCLIC) in airway epithelial cells [J. D. McCann, J. Matsuda, M. Garcia, G. Kaczorowski, and M. J. Welsh. Am. J. Physiol 258 (Lung Cell. Mol. Physiol. 2): L334-L342, 1990]. To determine whether the KCLIC channel is a basolateral membrane channel and to understand its role in Cl- secretion, we studied airway epithelial cells grown on permeable supports. When cells were stimulated with A23187, charybdotoxin (ChTX) inhibited Cl- secretion and 86Rb efflux at the same concentrations, indicating that the KCLIC channel is required for Ca2(+)-stimulated Cl- secretion. We also investigated the function of K+ channels in adenosine 3',5'-cyclic monophosphate-stimulated secretion. Addition of isoproterenol caused a biphasic increase in Cl- secretion; the time course of the transient component correlated with the time course of the isoproterenol-induced increase in Ca2+ concentration [( Ca2+]c). ChTX inhibited the transient component, but not the prolonged component of secretion; Ba2+ inhibited the sustained component. These results suggest that when cells are grown on permeable supports isoproterenol-induced secretion depends on activation of two types of K+ channel: the KCLIC channel that is stimulated initially and a ChTX-insensitive K+ channel that is stimulated during sustained secretion. This conclusion was supported by measurement of 86Rb efflux from cell monolayers

Stretch-activated channels in airway epithelial cells

1993 ◽  
Vol 265 (5) ◽  
pp. C1306-C1318 ◽  
Author(s):  
Y. K. Kim ◽  
E. R. Dirksen ◽  
M. J. Sanderson
Keyword(s):  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Reversal Potential ◽  
Airway Epithelial Cells ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Airway Epithelial ◽  
Open Probability ◽  
Open States ◽  
Slow Time Constant

Two type of stretch-activated (SA) ion channels were identified in the basolateral membrane of isolated rabbit airway epithelial cells by patch-clamp techniques. Pressure activation and deactivation of one channel, which had a conductance of 29 pS, occurred after a delay of approximately 20-30 s. The open probability of this delayed stretch-activated (DSA) channel was increased from < 0.01 to 0.45 at 50 mmHg of suction. The reversal potential of the DSA channel, calculated from the pipette potential at which membrane currents reversed [-31.3 +/- 3.6 (SD) mV] and the resting membrane potential (-27.8 +/- 3.3 mV) was +3.5 +/- 3.3 mV. None of the equilibrium potentials of the ions used were similar to the calculated reversal potential of the DSA channel, suggesting that this channel is nonselective for cations. The DSA channel gating behavior was characterized by bursts of rapid transitions between open and closed states. The distribution of the open and closed times revealed that this gating behavior could be fitted with two open states and two closed states. Only the slow time constant of the closed state was decreased by suction. The second SA channel was selective for K+ and had a conductance of 65 pS but a long delay was not associated with the pressure sensitivity of this channel. The open probability of the K(+)-selective SA channel was increased from < 0.01 to 0.30 by 50 mmHg of suction. The K(+)-selective SA channel was distinct from the well-characterized basolateral K+ channel.

Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents

1994 ◽  
Vol 266 (5) ◽  
pp. C1464-C1477 ◽  
Author(s):  
E. M. Schwiebert ◽  
T. Flotte ◽  
G. R. Cutting ◽  
W. B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Wild Type ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Cl Channel ◽  
Cl Channels

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

scholarly journals Expression of intermediate-conductance, Ca2+-activated K+ channel (KCNN4) in H441 human distal airway epithelial cells

2006 ◽  
Vol 291 (5) ◽  
pp. L957-L965 ◽  
Author(s):  
S. M. Wilson ◽  
S. G. Brown ◽  
N. McTavish ◽  
R. P. McNeill ◽  
E. M. Husband ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Conductance ◽  
Airway Epithelial Cells ◽  
K Channel ◽  
Channel Blockers ◽  
Resting Cells ◽  
Airway Epithelial ◽  
Distal Airway

Electrophysiological studies of H441 human distal airway epithelial cells showed that thapsigargin caused a Ca2+-dependent increase in membrane conductance ( GTot) and hyperpolarization of membrane potential ( Vm). These effects reflected a rapid rise in cellular K+ conductance ( GK) and a slow fall in amiloride-sensitive Na+ conductance ( GNa). The increase in GTot was antagonized by Ba2+, a nonselective K+ channel blocker, and abolished by clotrimazole, a KCNN4 inhibitor, but unaffected by other selective K+ channel blockers. Moreover, 1-ethyl-2-benzimidazolinone (1-EBIO), which is known to activate KCNN4, increased GK with no effect on GNa. RT-PCR-based analyses confirmed expression of mRNA encoding KCNN4 and suggested that two related K+ channels (KCNN1 and KCNMA1) were absent. Subsequent studies showed that 1-EBIO stimulates Na+ transport in polarized monolayers without affecting intracellular Ca2+ concentration ([Ca2+]i), suggesting that the activity of KCNN4 might influence the rate of Na+ absorption by contributing to GK. Transient expression of KCNN4 cloned from H441 cells conferred a Ca2+- and 1-EBIO-sensitive K+ conductance on Chinese hamster ovary cells, but this channel was inactive when [Ca2+]i was <0.2 μM. Subsequent studies of amiloride-treated H441 cells showed that clotrimazole had no effect on Vm despite clear depolarizations in response to increased extracellular K+ concentration ([K+]o). These findings thus indicate that KCNN4 does not contribute to Vm in unstimulated cells. The present data thus establish that H441 cells express KCNN4 and highlight the importance of GK to the control of Na+ absorption, but, because KCNN4 is quiescent in resting cells, this channel cannot contribute to resting GK or influence basal Na+ absorption.

scholarly journals P. aeruginosa Induced Lipid Peroxidation Causes Ferroptotic Cell Death in Airways

2021 ◽  
Vol 55 (5) ◽  
pp. 590-604
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Phospholipid Scramblase ◽  
Human Airway ◽  
Cl Channel ◽  
Human Airway Epithelial Cells

BACKGROUND/AIMS: Oxidative stress and infections by Pseudomonas aeruginosa (P. aeruginosa) are prominent in lungs of patients suffering from cystic fibrosis (CF). METHODS: The present study examines effects of P. aeruginosa on lipid peroxidation in human and mouse lungs, and cell death induced by P. aeruginosa in human airway epithelial cells. The role of the Ca2+ activated Cl- channel TMEM16A, the phospholipid scramblase TMEM16F, and the CFTR Cl- channel for ferroptotic cell death is examined. RESULTS: Lipid peroxidation was detected in human CF lungs, which correlated with bacterial infection. In vivo inoculation with P. aeruginosa or Staphylococcus aureus (S. aureus) induced lipid peroxidation in lungs of mice lacking expression of CFTR, and in lungs of wild type animals. Incubation of CFBE human airway epithelial cells with P. aeruginosa induced an increase in reactive oxygen species (ROS), causing lipid peroxidation and cell death independent of expression of wt-CFTR or F508del-CFTR. Knockdown of TMEM16A attenuated P. aeruginosa induced cell death. Antioxidants such as coenzyme Q10 and idebenone as well as the inhibitor of ferroptosis, ferrostatin-1, inhibited P. aeruginosa-induced cell death. CFBE cells expressing wtCFTR, but not F508del-CFTR, activated a basal Cl- conductance upon exposure to P. aeruginosa, which was caused by an increase in intracellular basal Ca2+ concentrations and activation of Ca2+-dependent adenylate cyclase. CONCLUSION: The data suggest an intrinsic pro-inflammatory phenotype in CF epithelial cells, while ferroptosis is observed in both non-CF and CF epithelial cells upon infection with P. aeruginosa. CF cells fail to activate fluid secretion in response to infection with P. aeruginosa. The use of antioxidants and inhibitors of ferroptosis is proposed as a treatment of pneumonia caused by infection with P. aeruginosa.

scholarly journals Inhibition of TMEM16A Cl − Channel by Fungus‐Derived Penicillide in Airway Epithelial Cells

2019 ◽  
Vol 33 (S1) ◽  
Author(s):  
Chantapol Yimnual ◽  
Saravut Satitsri ◽  
Vatcharin Rukachaisirikul ◽  
Chatchai Muanprasat
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cl Channel
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