Identification of a highly conserved sequence at the N-terminus of the epithelial Na + channel α subunit involved in gating

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7–9 days, the abundance of two forms of the α-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the β-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the γ-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in α- and γ-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in α- and γ- but not β-subunits when animals were treated with exogenous aldosterone, and in β- and γ- but not α-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney.

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The Epithelial Na+Channel Is Inhibited by a Peptide Derived from Proteolytic Processing of Its α Subunit

Journal of Biological Chemistry ◽

10.1074/jbc.m604109200 ◽

2006 ◽

Vol 281 (27) ◽

pp. 18901-18907 ◽

Cited By ~ 103

Author(s):

Marcelo D. Carattino ◽

Shaohu Sheng ◽

James B. Bruns ◽

Joseph M. Pilewski ◽

Rebecca P. Hughey ◽

...

Keyword(s):

Proteolytic Processing ◽

Na Channel ◽

Α Subunit ◽

Epithelial Na Channel

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Glycosylated extracellular asparagines of the α‐subunit provide a connection to the extracellular matrix and facilitate the shear force‐dependent activation of the human epithelial Na + ‐channel (ENaC)

The FASEB Journal ◽

10.1096/fasebj.29.1_supplement.845.1 ◽

2015 ◽

Vol 29 (S1) ◽

Author(s):

Fenja Knoepp ◽

Pawel Szczesniak ◽

Mike Althaus ◽

Martin Fronius

Keyword(s):

Extracellular Matrix ◽

Shear Force ◽

Na Channel ◽

Α Subunit ◽

Epithelial Na Channel

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Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its α subunit

Journal of Clinical Investigation ◽

10.1172/jci6821 ◽

1999 ◽

Vol 104 (7) ◽

pp. 967-974 ◽

Cited By ~ 77

Author(s):

Olivier Bonny ◽

Ahmed Chraibi ◽

Jan Loffing ◽

Nicole Fowler Jaeger ◽

Stefan Gründer ◽

...

Keyword(s):

Functional Expression ◽

Na Channel ◽

Type I ◽

Α Subunit ◽

Epithelial Na Channel ◽

Pseudohypoaldosteronism Type

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Identification of an Amiloride Binding Domain within the α-Subunit of the Epithelial Na+Channel

Journal of Biological Chemistry ◽

10.1074/jbc.272.34.21075 ◽

1997 ◽

Vol 272 (34) ◽

pp. 21075-21083 ◽

Cited By ~ 58

Author(s):

Iskander I. Ismailov ◽

Thomas Kieber-Emmons ◽

Chaomei Lin ◽

Bakhram K. Berdiev ◽

Vadim Gh. Shlyonsky ◽

...

Keyword(s):

Binding Domain ◽

Na Channel ◽

Α Subunit ◽

Epithelial Na Channel

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Location and function of the epithelial Na channel in the cochlea

AJP Renal Physiology ◽

10.1152/ajprenal.2001.280.2.f214 ◽

2001 ◽

Vol 280 (2) ◽

pp. F214-F222 ◽

Cited By ~ 37

Author(s):

Vincent Couloigner ◽

Michel Fay ◽

Sabri Djelidi ◽

Nicolette Farman ◽

Brigitte Escoubet ◽

...

Keyword(s):

Stria Vascularis ◽

Spiral Ganglion ◽

Endocochlear Potential ◽

Na Channel ◽

Spiral Ligament ◽

Α Subunit ◽

Subunit Mrna ◽

Spiral Limbus ◽

Epithelial Na Channel

In the cochlea, endolymph is a K-rich and Na-poor fluid. The purpose of the present study was to check the presence and to assess the role of epithelial Na channel (ENaC) in this organ. α-, β-, and γ-ENaC subunit mRNA, and proteins were detected in rat cochlea by RT-PCR and Western blot. α-ENaC subunit mRNA was localized by in situ hybridization in both epithelial (stria vascularis, spiral prominence, spiral limbus) and nonepithelial structures (spiral ligament, spiral ganglion). The α-ENaC-positive tissues were also positive for β-subunit mRNA (except spiral ganglion) or for γ-subunit mRNA (spiral limbus, spiral ligament, and spiral ganglion), but the signals of β- and γ-subunits were weaker than those observed for α-subunit. In vivo, the endocochlear potential was recorded in guinea pigs under normoxic and hypoxic conditions after endolymphatic perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear potential when Na-rich but not when K-rich solutions were perfused. In conclusion, ENaC subunits are expressed in epithelial and nonepithelial cochlear structures. One of its functions is probably to maintain the low concentration of Na in endolymph.

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Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the α Subunit of the Epithelial Na+Channel

Journal of Biological Chemistry ◽

10.1074/jbc.274.14.9648 ◽

1999 ◽

Vol 274 (14) ◽

pp. 9648-9655 ◽

Cited By ~ 21

Author(s):

Thomas Kieber-Emmons ◽

Chaomei Lin ◽

Mary H. Foster ◽

Thomas R. Kleyman

Keyword(s):

Binding Domain ◽

Na Channel ◽

Α Subunit ◽

Antiidiotypic Antibody ◽

Epithelial Na Channel

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On the Molecular Basis of Ion Permeation in the Epithelial Na+ Channel

The Journal of General Physiology ◽

10.1085/jgp.114.1.13 ◽

1999 ◽

Vol 114 (1) ◽

pp. 13-30 ◽

Cited By ~ 94

Author(s):

Stephan Kellenberger ◽

Nicole Hoffmann-Pochon ◽

Ivan Gautschi ◽

Estelle Schneeberger ◽

Laurent Schild

Keyword(s):

Amino Acid ◽

Single Channel ◽

Kinetic Property ◽

Selectivity Filter ◽

Na Channel ◽

Amino Acid Residues ◽

Discrete State ◽

Α Subunit ◽

Α Helix ◽

Epithelial Na Channel

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two α, one β, and one γ homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of β and γ subunits, which precedes the second putative transmembrane α helix (M2). A residue in the α subunit (αS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of α, β, and γ subunits (αG587, βG529, γS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of βG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported αS589 mutations. We conclude that the residues αG587 to αS589 and homologous residues in the β and γ subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

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