International Union of Basic and Clinical Pharmacology. XCI. Structure, Function, and Pharmacology of Acid-Sensing Ion Channels and the Epithelial Na+ Channel

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 ∼ 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.

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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.

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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.

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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.

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

Physiology ◽

10.1152/physiologyonline.1996.11.5.195 ◽

1996 ◽

Vol 11 (5) ◽

pp. 195-201

Author(s):

CM Canessa

Keyword(s):

Ion Channels ◽

Apical Membrane ◽

Na Channel ◽

Full Activity ◽

New Family ◽

Epithelial Na Channel ◽

Tight Epithelium

The epithelial Na+ channel (ENaC) in the apical membrane of tight epithelium represents the first member of a new family of ion channels. The channel is formed by the association of three homologous subunits, a-, b-, and g-ENaC, that functionally complement to give full activity to the channel complex.

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