scholarly journals CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1858
Author(s):  
Waheed Shabbir ◽  
Nermina Topcagic ◽  
Mohammed Aufy ◽  
Murat Oz
Keyword(s):  
A549 Cells ◽  
Na Channel ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Knock Down ◽  
Whole Cell Patch Clamp ◽  
Glycosylation Sites ◽  
Epithelial Na Channel

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

scholarly journals Mechanical activation of epithelial Na+ channel relies on an interdependent activity of the extracellular matrix and extracellular N-glycans of αENaC

2017 ◽  
Author(s):  
Fenja Knoepp ◽  
Zoe Ashley ◽  
Daniel Barth ◽  
Marina Kazantseva ◽  
Pawel P. Szczesniak ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ion Channels ◽  
Shear Force ◽  
Na Channel ◽  
Pressure Regulation ◽  
Mechanical Environment ◽  
Mechanosensitive Ion Channels ◽  
Glycosylation Sites ◽  
Epithelial Na Channel

AbstractMechanotransduction describes how cells perceive their mechanical environment and mechanosensitive ion channels are important for this process. ENaC (epithelial Na+ channel)/DEG (degenerin) proteins form mechanosensitive ion channels and it is hypothesized their interaction with the extracellular matrix (ECM) via ‘tethers’ is required for mechanotransduction. Channels formed by vertebrate α, β and γ ENaC proteins are activated by shear force (SF) and mediate electrolyte/fluid-homeostasis and blood pressure regulation. Here, we report an interdependent activity of ENaC and the ECM that mediates SF effects in murine arteries and heterologously expressed channels. Furthermore, replacement of conserved extracellular N-glycosylated asparagines of αENaC decreased the SF response indicating that the attached N-glycans provide a connection to the ECM. Insertion of N-glycosylation sites into a channel subunit, innately lacking these motifs, increased its SF response. These experiments confirm an interdependent channel/ECM activity of mechanosensitive ENaC channel and highlight the role of channel N-glycans as new constituents for the translation of mechanical force into cellular signals.

scholarly journals Expression and function of KV2-containing channels in human urinary bladder smooth muscle

2012 ◽  
Vol 302 (11) ◽  
pp. C1599-C1608 ◽  
Author(s):  
Kiril L. Hristov ◽  
Muyan Chen ◽  
Serge A. Y. Afeli ◽  
Qiuping Cheng ◽  
Eric S. Rovner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Electrical Field Stimulation ◽  
Bladder Smooth Muscle ◽  
Rt Pcr ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

The functional role of the voltage-gated K+ (KV) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of KV2.1, KV2.2, and the electrically silent KV9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of KV2.1, KV2.2, and KV4.2 homotetrameric channels and of KV2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca2+ imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of KV2.1, KV2.2, and KV9.3 (but not KV4.2) channel subunits in human isolated DSM cells. KV2.1 and KV2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced KV current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca2+ level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5–30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive KV2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

scholarly journals Inhibition of cardiac Na+ currents by isoproterenol

1990 ◽  
Vol 258 (4) ◽  
pp. H977-H982 ◽  
Author(s):  
B. Schubert ◽  
A. M. Vandongen ◽  
G. E. Kirsch ◽  
A. M. Brown
Keyword(s):  
Patch Clamp ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Na Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Voltage Dependent

The mechanism by which the beta-adrenergic agonist isoproterenol (ISO) modulates voltage-dependent cardiac Na+ currents (INa) was studied in single ventricular myocytes of neonatal rat using the gigaseal patch-clamp technique. ISO inhibited INa reversibly, making the effect readily distinguishable from the monotonic decrease of INa caused by the shift in gating that customarily occurs during whole cell patch-clamp experiments (E. Fenwick, A. Marty, and E. Neher, J. Physiol. Lond. 331: 599-635, 1982; and J. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. Lond. 356: 565-585, 1984). The inhibition was biphasic, having fast and slow components, and was voltage-dependent, being more pronounced at depolarized potentials. In whole cell experiments the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) congener 8-bromo-cAMP reduced INa. In cell-free inside-out patches with ISO present in the pipette, guanosine 5'-triphosphate (GTP) applied to the inner side of the membrane patch inhibited single Na+ channel activity. This inhibition could be partly reversed by hyperpolarizing prepulses. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) greatly reduced the probability of single Na+ channel currents in a Mg2(+)-dependent manner. We propose that ISO inhibits cardiac Na+ channels via the guanine nucleotide binding, signal-transducing G protein that acts through both direct (membrane delimited) and indirect (cytoplasmic) pathways.

Acute effects of repetitive depolarization on sodium current in chick myocytes

1991 ◽  
Vol 260 (6) ◽  
pp. H1810-H1818
Author(s):  
M. R. Gold ◽  
G. R. Strichartz
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Complete Recovery ◽  
Acute Effects ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

scholarly journals Regulation of the Epithelial Na+ Channel by Membrane Tension

1998 ◽  
Vol 112 (2) ◽  
pp. 97-111 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Muthangi Subramanyam
Keyword(s):  
Cytochalasin B ◽  
Single Channel ◽  
Direct Injection ◽  
Expression System ◽  
Cell Swelling ◽  
Na Channel ◽  
Bathing Solution ◽  
Membrane Tension ◽  
Whole Cell ◽  
Epithelial Na Channel

The sensitivity of αβγ rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

scholarly journals Surface Expression of Epithelial Na Channel Protein in Rat Kidney

2008 ◽  
Vol 131 (6) ◽  
pp. 617-627 ◽  
Author(s):  
Gustavo Frindt ◽  
Zuhal Ergonul ◽  
Lawrence G. Palmer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Surface Expression ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Renal Cells ◽  
Patch Clamp Recording ◽  
Membrane Fractionation ◽  
Whole Cell Patch Clamp ◽  
Epithelial Na Channel

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.

scholarly journals Differential Interactions of Na+ Channel Toxins with T-type Ca2+ Channels

2008 ◽  
Vol 132 (1) ◽  
pp. 101-113 ◽  
Author(s):  
Hui Sun ◽  
Diego Varela ◽  
Denis Chartier ◽  
Peter C. Ruben ◽  
Stanley Nattel ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Low Voltage ◽  
Parallel Evolution ◽  
Na Channel ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
The Right ◽  
Ca2 Channels

Two types of voltage-dependent Ca2+ channels have been identified in heart: high (ICaL) and low (ICaT) voltage-activated Ca2+ channels. In guinea pig ventricular myocytes, low voltage–activated inward current consists of ICaT and a tetrodotoxin (TTX)-sensitive ICa component (ICa(TTX)). In this study, we reexamined the nature of low-threshold ICa in dog atrium, as well as whether it is affected by Na+ channel toxins. Ca2+ currents were recorded using the whole-cell patch clamp technique. In the absence of external Na+, a transient inward current activated near −50 mV, peaked at −30 mV, and reversed around +40 mV (HP = −90 mV). It was unaffected by 30 μM TTX or micromolar concentrations of external Na+, but was inhibited by 50 μM Ni2+ (by ∼90%) or 5 μM mibefradil (by ∼50%), consistent with the reported properties of ICaT. Addition of 30 μM TTX in the presence of Ni2+ increased the current approximately fourfold (41% of control), and shifted the dose–response curve of Ni2+ block to the right (IC50 from 7.6 to 30 μM). Saxitoxin (STX) at 1 μM abolished the current left in 50 μM Ni2+. In the absence of Ni2+, STX potently blocked ICaT (EC50 = 185 nM) and modestly reduced ICaL (EC50 = 1.6 μM). While TTX produced no direct effect on ICaT elicited by expression of hCaV3.1 and hCaV3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni2+ (IC50 increased to 550 μM Ni2+ for CaV3.1 and 15 μM Ni2+ for CaV3.2); in contrast, 30 μM TTX directly inhibited hCaV3.3-induced ICaT and the addition of 750 μM Ni2+ to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni2+ alone. 1 μM STX directly inhibited CaV3.1-, CaV3.2-, and CaV3.3-mediated ICaT but did not enhance the ability of Ni2+ to block these currents. These findings provide important new implications for our understanding of structure–function relationships of ICaT in heart, and further extend the hypothesis of a parallel evolution of Na+ and Ca2+ channels from an ancestor with common structural motifs.

Activation of Phosphatidylinositol-Linked Novel D1 Dopamine Receptors Inhibits High-Voltage-Activated Ca2+ Currents in Primary Cultured Striatal Neurons

2009 ◽  
Vol 101 (5) ◽  
pp. 2230-2238 ◽  
Author(s):  
Li-Qun Ma ◽  
Chao Liu ◽  
Fang Wang ◽  
Na Xie ◽  
Jun Gu ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
High Voltage ◽  
Dependent Manner ◽  
Striatal Neurons ◽  
Inhibitory Effects ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Intracellular Ca ◽  
Dose Dependent

Recent evidences indicate the existence of a putative novel phosphatidylinositol (PI)-linked D1 dopamine receptor that mediates excellent anti-Parkinsonian but less severe dyskinesia action. To further understand the basic physiological function of this receptor in brain, the effects of a PI-linked D1 dopamine receptor-selective agonist 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) on high-voltage activated (HVA) Ca2+ currents in primary cultured striatal neurons were investigated by whole cell patch-clamp technique. The results indicated that stimulation by SKF83959 induced an inhibition of HVA Ca2+ currents in a dose-dependent manner in substance-P (SP)-immunoreactive striatal neurons. Application of D1 receptor, but not D2, α1 adrenergic, 5-HT receptor, or cholinoceptor antagonist prevented SKF83959-induced reduction, indicating that a D1 receptor-mediated event assumed via PI-linked D1 receptor. SKF83959-induced inhibitory modulation was mediated by activation of phospholipase C (PLC), mobilization of intracellular Ca2+ stores and activation of calcineurin. Furthermore, the inhibitory effects were attenuated significantly by the L-type calcium channel antagonist nifedipine, suggesting that L-type calcium channels involved in the regulation induced by SKF83959. These findings may help to further understand the functional role of the PI-linked dopamine receptor in brain.

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