Faculty Opinions recommendation of Acute cholesterol-induced anti-natriuretic effects: role of epithelial Na+ channel activity, protein levels, and processing.

2011 ◽  
Author(s):  
Franz-Xaver Beck
Keyword(s):  
Na Channel ◽  
Channel Activity ◽  
Protein Levels ◽  
Epithelial Na Channel

In vitro phosphorylation of COOH termini of the epithelial Na+ channel and its effects on channel activity inXenopus oocytes

2001 ◽  
Vol 280 (6) ◽  
pp. F1030-F1036 ◽  
Author(s):  
Alexander Chigaev ◽  
Gang Lu ◽  
Haikun Shi ◽  
Carol Asher ◽  
Rong Xu ◽  
...  
Keyword(s):  
Rat Colon ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Glutathione S Transferase ◽  
Single Well ◽  
Py Motif ◽  
Epithelial Na Channel

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the γ-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on β(S620) was phosphorylated as well. The possible role of γT630 and βS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na+ and had a high basal Na+ current. Thus mutating γT630 or βS620 may limit the maximal channel activity achieved by a combination of sgk and low Na+.

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 Acute effects of aldosterone on the epithelial Na channel in rat kidney

2015 ◽  
Vol 308 (6) ◽  
pp. F572-F578 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Rat Kidney ◽  
Surface Protein ◽  
Early Response ◽  
Surface Expression ◽  
Na Channel ◽  
Channel Activity ◽  
Acute Effects ◽  
Chronic Stimulation ◽  
Epithelial Na Channels ◽  
Epithelial Na Channel

The acute effects of aldosterone administration on epithelial Na channels (ENaC) in rat kidney were examined using electrophysiology and immunodetection. Animals received a single injection of aldosterone (20 μg/kg body wt), which reduced Na excretion over the next 3 h. Channel activity was assessed in principal cells of cortical collecting ducts as amiloride-sensitive whole cell clamp current ( INa). INa averaged 100 pA/cell, 20–30% of that reported for the same preparation under conditions of chronic stimulation. INa was negligible in control animals that did not receive hormone. The acute physiological response correlated with changes in ENaC processing and trafficking. These effects included increases in the cleaved forms of α-ENaC and γ-ENaC, assessed by Western blot, and increases in the surface expression of β-ENaC and γ-ENaC measured after surface protein biotinylation. These changes were qualitatively and quantitatively similar to those of chronic stimulation. This suggests that altered trafficking to or from the apical membrane is an early response to the hormone and that later increases in channel activity require stimulation of channels residing at the surface.

scholarly journals Epithelial Na+ channel activation and processing in mice lacking SGK1

2008 ◽  
Vol 294 (6) ◽  
pp. F1298-F1305 ◽  
Author(s):  
Geza Fejes-Tóth ◽  
Gustavo Frindt ◽  
Aniko Náray-Fejes-Tóth ◽  
Lawrence G. Palmer
Keyword(s):  
Full Length ◽  
Na Channel ◽  
Channel Activity ◽  
Salt Wasting ◽  
Optimal Processing ◽  
Collecting Ducts ◽  
Cell Current ◽  
Dependent Protein ◽  
Knockout Animals ◽  
Epithelial Na Channel

Amiloride-sensitive Na+ channel activity was examined in the cortical collecting ducts of a mouse line (SGK1−/−) deficient in the serum- and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial Na+ channel (ENaC) protein. Neither SGK1−/− mice nor paired SGK1+/+ animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current ( INa), under control conditions with standard chow. Administration of aldosterone (0.5 μg/h via osmotic minipump for 7 days) increased INa to a similar extent in SGK1+/+ (378 ± 61 pA/cell at −100 mV) and in SGK1−/− (350 ± 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of γ-ENaC, was more pronounced in SGK+/+ mice. The SGK1−/− animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, INa was enhanced more in SGK1−/− (94 ± 14 pA/cell) than in SGK+/+ (23 ± 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length γ-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of Na+-Cl− cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone.

scholarly journals Regulation of renal Na transporters in response to dietary K

2018 ◽  
Vol 315 (4) ◽  
pp. F1032-F1041 ◽  
Author(s):  
Lei Yang ◽  
Shuhua Xu ◽  
Xiaoyun Guo ◽  
Shinichi Uchida ◽  
Alan M. Weinstein ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Time Course ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Protein Levels ◽  
Phosphorylated Form ◽  
High K ◽  
Enhanced Expression ◽  
Males And Females ◽  
Epithelial Na Channel

Changes in the expression of Na transport proteins were measured in the kidneys of mice with increased dietary K intake for 1 wk. The epithelial Na channel (ENaC) was upregulated, with enhanced expression of full-length and cleaved forms of α-ENaC and cleaved γ-ENaC. At the same time, the amount of the NaCl cotransporter NCC and its phosphorylated form decreased by ~50% and ~80%, respectively. The expression of the phosphorylated form of the Na-K-2Cl cotransporter NKCC2 also decreased, despite an increase in overall protein content. The effect was stronger in males (80%) than in females (40%). This implies that less Na+ is reabsorbed in the thick ascending limb of Henle’s loop and distal convoluted tubule along with Cl−, whereas more is reabsorbed in the aldosterone-sensitive distal nephron in exchange for secreted K+. The abundance of the proximal tubule Na/H exchanger NHE3 decreased by ~40%, with similar effects in males and females. Time-course studies indicated that NCC and NHE3 proteins decreased progressively over 7 days on a high-K diet. Expression of mRNA encoding these proteins increased, implying that the decreased protein levels resulted from decreased rates of synthesis or increased rates of degradation. The potential importance of changes in NHE3, NKCC2, and NCC in promoting K+ excretion was assessed with a mathematical model. Simulations indicated that decreased NHE3 produced the largest effect. Regulation of proximal tubule Na+ transport may play a significant role in achieving K homeostasis.

scholarly journals Impaired epithelial Na+ channel activity contributes to cystogenesis and development of autosomal recessive polycystic kidney disease in PCK rats

2014 ◽  
Vol 77 (1) ◽  
pp. 64-69 ◽  
Author(s):  
Tengis S. Pavlov ◽  
Vladislav Levchenko ◽  
Daria V. Ilatovskaya ◽  
Oleg Palygin ◽  
Alexander Staruschenko
Keyword(s):  
Kidney Disease ◽  
Polycystic Kidney Disease ◽  
Autosomal Recessive ◽  
Na Channel ◽  
Channel Activity ◽  
Polycystic Kidney ◽  
Epithelial Na Channel

scholarly journals Oxygen regulation of the epithelial Na channel in the collecting duct

2011 ◽  
Vol 300 (2) ◽  
pp. F412-F424 ◽  
Author(s):  
Russell F. Husted ◽  
Hongyan Lu ◽  
Rita D. Sigmund ◽  
John B. Stokes
Keyword(s):  
Kinase Activity ◽  
Collecting Duct ◽  
Hypoxia Inducible Factor ◽  
Na Channel ◽  
Mrna Levels ◽  
Apical Surface ◽  
Amp Kinase ◽  
Na Transport ◽  
Protein Levels ◽  
Epithelial Na Channel

The Po2 within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing Po2 on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O2 concentration from 20 to 8% decreased ENaC activity by 40%; increasing O2 content to 40% increased ENaC activity by 50%. The O2 effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O2 medium. Corticosteroids increased ENaC activity at each O2 concentration; there was no interaction. The pathways for O2 and steroid regulation of ENaC are different since O2 did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2–45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O2 appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O2 concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O2 induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O2; both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.

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