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 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 Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers

1995 ◽  
Vol 268 (6) ◽  
pp. C1450-C1459 ◽  
Author(s):  
M. S. Awayda ◽  
I. I. Ismailov ◽  
B. K. Berdiev ◽  
D. J. Benos
Keyword(s):  
Hydrostatic Pressure ◽  
Pressure Gradient ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Stretch Activation ◽  
Hydrostatic Pressure Gradient ◽  
Epithelial Na Channel

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.

scholarly journals The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

1998 ◽  
Vol 141 (1) ◽  
pp. 281-286 ◽  
Author(s):  
Albrecht Lepple-Wienhues ◽  
Ildikò Szabò ◽  
Tilmann Laun ◽  
Nubia Kristen Kaba ◽  
Erich Gulbins ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Tyrosine Kinase ◽  
Single Channel ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Osmotic Swelling ◽  
Whole Cell ◽  
Herbimycin A ◽  
Unitary Conductance ◽  
Human T Cell

Osmotic cell swelling activates Cl− channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl−swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl−swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl−swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl−swell in lymphocytes via the tyrosine kinase p56lck.

Functional Reconstitution of the Human Epithelial Na+ Channel in a Mammalian Expression System

Ion Channels ◽  
2006 ◽  
pp. 3-13
Author(s):  
Alexander Staruschenko ◽  
Rachell E. Booth ◽  
Oleh Pochynyuk ◽  
James D. Stockand ◽  
Qiusheng Tong
Keyword(s):  
Expression System ◽  
Na Channel ◽  
Mammalian Expression ◽  
Mammalian Expression System ◽  
Epithelial Na Channel

Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells

1991 ◽  
Vol 261 (4) ◽  
pp. C658-C674 ◽  
Author(s):  
C. K. Solc ◽  
J. J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Current Density ◽  
Single Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Whole Cell ◽  
Cl Channels ◽  
Single Channel Currents

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells

1997 ◽  
Vol 272 (2) ◽  
pp. C466-C475 ◽  
Author(s):  
H. F. Cantiello ◽  
G. R. Jackson ◽  
A. G. Prat ◽  
J. L. Gazley ◽  
J. N. Forrest ◽  
...  
Keyword(s):  
Single Channel ◽  
Primary Cultures ◽  
Disulfonic Acid ◽  
Rectal Gland ◽  
Bathing Solution ◽  
Whole Cell ◽  
Extracellular Milieu ◽  
Conductive Pathway ◽  
Shark Rectal Gland ◽  
Camp Stimulation

The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 microM) and glibenclamide (400 microM). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents in excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.

scholarly journals Protein kinase regulation of a cloned epithelial Na+ channel.

1996 ◽  
Vol 108 (1) ◽  
pp. 49-65 ◽  
Author(s):  
M S Awayda ◽  
I I Ismailov ◽  
B K Berdiev ◽  
C M Fuller ◽  
D J Benos
Keyword(s):  
Protein Kinase ◽  
Lipid Bilayers ◽  
Xenopus Oocytes ◽  
Cytochalasin B ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Open Probability ◽  
Alpha Beta ◽  
Epithelial Na Channel ◽  
Stimulation Of

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.

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