What Is New About the Structure of the Epithelial Na+ Channel ?

Epithelial Na+ channel (ENaC) participates in renal epithelial Na+ reabsorption, controlling blood pressure. Aldosterone and insulin elevate blood pressure by increasing the ENaC-mediated Na+ reabsorption. However, little information is available on the interactive action of aldosterone and insulin on the ENaC-mediated Na+ reabsorption. In the present study, we tried to clarify if insulin would modify the aldosterone action on the ENaC-mediated Na+ reabsorption from a viewpoint of intracellular ENaC trafficking. We measured the ENaC-mediated Na+ transport as short-circuit currents using a four-state mathematical ENaC trafficking model in renal A6 epithelial cells with or without aldosterone treatment under the insulin-stimulated and -unstimulated conditions. We found that: (A) under the insulin-stimulated condition, aldosterone treatment (1 µM for 20 h) significantly elevated the ENaC insertion rate to the apical membrane ( k I ) 3.3-fold and the ENaC recycling rate ( k R ) 2.0-fold, but diminished the ENaC degradation rate ( k D ) 0.7-fold without any significant effect on the ENaC endocytotic rate ( k E ); (B) under the insulin-unstimulated condition, aldosterone treatment decreased k E 0.5-fold and increased k R 1.4-fold, without any significant effect on k I or k D . Thus, the present study indicates that: (1) insulin masks the well-known inhibitory action of aldosterone on the ENaC endocytotic rate; (2) insulin induces a stimulatory action of aldosterone on ENaC apical insertion and an inhibitory action of aldosterone on ENaC degradation; (3) insulin enhances the aldosterone action on ENaC recycling; (4) insulin has a more effective action on diminution of ENaC endocytosis than aldosterone.

Download Full-text

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

10.1101/102756 ◽

2017 ◽

Cited By ~ 3

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.

Download Full-text

Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel

The Journal of General Physiology ◽

10.1085/jgp.200709800 ◽

2007 ◽

Vol 130 (4) ◽

pp. 399-413 ◽

Cited By ~ 62

Author(s):

Oleh Pochynyuk ◽

Qiusheng Tong ◽

Jorge Medina ◽

Alain Vandewalle ◽

Alexander Staruschenko ◽

...

Keyword(s):

Ion Channels ◽

Single Channel ◽

Second Messengers ◽

Transmembrane Domains ◽

Effector Proteins ◽

Na Channel ◽

Open Probability ◽

Single Channel Analysis ◽

Molecular Determinants ◽

Epithelial Na Channel

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

Download Full-text

Effects of dietary K on cell-surface expression of renal ion channels and transporters

AJP Renal Physiology ◽

10.1152/ajprenal.00323.2010 ◽

2010 ◽

Vol 299 (4) ◽

pp. F890-F897 ◽

Cited By ~ 79

Author(s):

Gustavo Frindt ◽

Lawrence G. Palmer

Keyword(s):

Ion Channels ◽

Surface Protein ◽

Surface Expression ◽

Cell Surface Expression ◽

Secretory Cells ◽

Na Channel ◽

Apical Surface ◽

High K ◽

Low K ◽

Epithelial Na Channel

Changes in apical surface expression of ion channels and transporters in the superficial rat renal cortex were assessed using biotinylation and immunoblotting during alterations in dietary K intake. A high-K diet increased, and a low-K diet decreased, both the overall and surface abundance of the β- and γ-subunits of the epithelial Na channel (ENaC). In the case of γ-ENaC, the effect was specific for the 65-kDa cleaved form of the protein. The overall amount of α-ENAC was also increased with increasing K intake. The total expression of the secretory K+ channels (ROMK) increased with a high-K diet and decreased with a low-K diet. The surface expression of ROMK increased with high K intake but was not significantly altered by a low-K diet. In contrast, the amounts of total and surface protein representing the thiazide-sensitive NaCl cotransporter (NCC) decreased with increasing K intake. We conclude that modulation of K+ secretion in response to changes in dietary K intake involves changes in apical K+ permeability through regulation of K+ channels and in driving force subsequent to alterations in both Na delivery to the distal nephron and Na+ uptake across the apical membrane of the K+ secretory cells.

Download Full-text

Membrane trafficking pathways regulating the epithelial Na+ channel

AJP Renal Physiology ◽

10.1152/ajprenal.00277.2019 ◽

2020 ◽

Vol 318 (1) ◽

pp. F1-F13 ◽

Cited By ~ 5

Author(s):

Adam W. Ware ◽

Sahib R. Rasulov ◽

Tanya T. Cheung ◽

J. Shaun Lott ◽

Fiona J McDonald

Keyword(s):

Cell Surface ◽

Blood Volume ◽

Membrane Trafficking ◽

Apical Membrane ◽

Na Channel ◽

Membrane Domain ◽

Multiple Forms ◽

Epithelial Na Channel

Renal Na+ reabsorption, facilitated by the epithelial Na+ channel (ENaC), is subject to multiple forms of control to ensure optimal body blood volume and pressure through altering both the ENaC population and activity at the cell surface. Here, the focus is on regulating the number of ENaCs present in the apical membrane domain through pathways of ENaC synthesis and targeting to the apical membrane as well as ENaC removal, recycling, and degradation. Finally, the mechanisms by which ENaC trafficking pathways are regulated are summarized.

Download Full-text

Single-channel behavior of a purified epithelial Na+ channel subunit that binds amiloride

AJP Cell Physiology ◽

10.1152/ajpcell.1992.263.5.c1111 ◽

1992 ◽

Vol 263 (5) ◽

pp. C1111-C1117 ◽

Cited By ~ 14

Author(s):

S. Sariban-Sohraby ◽

M. Abramow ◽

R. S. Fisher

Keyword(s):

Single Channel ◽

Apical Membrane ◽

Na Channel ◽

Na Transport ◽

Patch Clamp Technique ◽

Transport Inhibitor ◽

Open Time ◽

The Mean ◽

Epithelial Na Channel ◽

Binding Subunit

The apical membrane of high electrical resistance epithelia, which is selectively permeable to Na+, plays an essential role in the maintenance of salt balance. Na+ entry from the apical fluid into the cells is mediated by amiloride-blockable Na(+)-specific channels. The channel protein, purified from both amphibian and mammalian sources, is composed of several subunits, only one of which the 150-kDa polypeptide, specifically binds the Na+ transport inhibitor amiloride. The goal of the present study was to investigate whether the isolated amiloride-binding subunit of the channel could conduct Na+. The patch-clamp technique was used to study the 150-kDa polypeptide incorporated into a lipid bilayer formed on the tip of a glass pipette. Unitary conductance jumps averaged 4.8 pS at 100 mM Na2HPO4. Open times ranged from 24 ms to several seconds. The channel spent most of the time in the closed state. Channel conductance and gating were independent of voltage between -60 and +100 mV. Amiloride (0.1 microM) decreased the mean open time of the channel by 98%. We conclude that the 150-kDa subunit of the amiloride-blockable Na+ channel conducts current and may be sufficient for the Na+ transport function of the whole channel.

Download Full-text