Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.

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Differential Regulation of the Epithelial Sodium Channel (ENaC) in Rat Kidney, Lung and Distal Colon in Response to Aldosterone.

Hypertension ◽

10.1161/hyp.36.suppl_1.724 ◽

2000 ◽

Vol 36 (suppl_1) ◽

pp. 724-724

Author(s):

Shyama M E Masilamani ◽

Gheun-Ho Kim ◽

Mark A Knepper

Keyword(s):

Sodium Channel ◽

Epithelial Sodium Channel ◽

Distal Colon ◽

Β Subunit ◽

Dietary Salt ◽

Α Subunit ◽

Salt Restriction ◽

Γ Subunit ◽

Dietary Salt Restriction ◽

Epithelial Sodium Channel Enac

P170 The mineralocorticoid hormone, aldosterone increases renal tubule Na absorption via increases in the protein abundances of the α-subunit of the epithelial sodium channel (ENaC) and the 70 kDa form of the γ- subunit of ENaC (JCI 104:R19-R23). This study assesses the affect of dietary salt restriction on the regulation of the epithelial sodium channel (ENaC) in the lung and distal colon, in addition to kidney, using semiquantitative immunoblotting. Rats were placed initially on either a control Na intake (0.02 meq/day), or a low Na intake (0.2 meq/day) for 10 days. The low salt treated rats demonstrated an increase in plasma aldosterone levels at day 10 (control = 0.78 + 0.32 nM; Na restricted = 3.50 + 1.30 nM). In kidney homogenates, there were marked increases in the band density of the α-subunit of ENaC (286 % of control) and the 70 kDa form of γ-subunit of ENaC (262 % of control), but no increase in the abundance of the β-subunit of ENaC. In lung homogenates, there was no significant change in the band densities of the α, β, or γ subunits of ENaC. In distal colon, there was an increase in the band density of the β-subunit of ENaC (311 % of control) and an increase in both the 85 kDa (2355% of control) and 70 kDa (843 % of control) form of the γ subunit of ENaC in response to dietary Na restriction. However, there was no significant difference in the band density of the α-subunit of ENaC. These findings demonstrate tissue specific regulation of the three subunits of ENaC in response to dietary salt restriction.

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Sac I identifies a biallelic polymorphism in the coding sequence of the gamma subunit of the epithelial sodium channel (ENaC): a candidate gene for hypertension

Clinical Genetics ◽

10.1111/j.1399-0004.1998.tb03705.x ◽

2008 ◽

Vol 54 (1) ◽

pp. 104-105 ◽

Cited By ~ 2

Author(s):

Rita Viaplana ◽

Esteban Poch ◽

Sergio Lario ◽

Josep Oriola ◽

Daniel González ◽

...

Keyword(s):

Sodium Channel ◽

Candidate Gene ◽

Epithelial Sodium Channel ◽

Gamma Subunit ◽

Coding Sequence ◽

Epithelial Sodium Channel Enac

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Structure of the human epithelial sodium channel by cryo-electron microscopy

eLife ◽

10.7554/elife.39340 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 47

Author(s):

Sigrid Noreng ◽

Arpita Bharadwaj ◽

Richard Posert ◽

Craig Yoshioka ◽

Isabelle Baconguis

Keyword(s):

Electron Microscopy ◽

Ion Channel ◽

Sodium Channel ◽

Conformational Changes ◽

Single Particle ◽

Transmembrane Domain ◽

Epithelial Sodium Channel ◽

Cryo Electron Microscopy ◽

Inhibitory Domain ◽

Epithelial Sodium Channel Enac

The epithelial sodium channel (ENaC), a member of the ENaC/DEG superfamily, regulates Na+ and water homeostasis. ENaCs assemble as heterotrimeric channels that harbor protease-sensitive domains critical for gating the channel. Here, we present the structure of human ENaC in the uncleaved state determined by single-particle cryo-electron microscopy. The ion channel is composed of a large extracellular domain and a narrow transmembrane domain. The structure reveals that ENaC assembles with a 1:1:1 stoichiometry of α:β:γ subunits arranged in a counter-clockwise manner. The shape of each subunit is reminiscent of a hand with key gating domains of a ‘finger’ and a ‘thumb.’ Wedged between these domains is the elusive protease-sensitive inhibitory domain poised to regulate conformational changes of the ‘finger’ and ‘thumb’; thus, the structure provides the first view of the architecture of inhibition of ENaC.

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Downregulation of epithelial sodium channel (ENaC) activity in human airway epithelia after low temperature incubation

BMJ Open Respiratory Research ◽

10.1136/bmjresp-2020-000861 ◽

2021 ◽

Vol 8 (1) ◽

pp. e000861

Author(s):

Sangya Yadav ◽

Ciaran A Shaughnessy ◽

Pamela L Zeitlin ◽

Preston E Bratcher

Keyword(s):

Low Temperature ◽

Sodium Channel ◽

Short Circuit ◽

Ussing Chamber ◽

Epithelial Sodium Channel ◽

Functional Expression ◽

Airway Epithelia ◽

Epithelial Cultures ◽

Epithelial Sodium Channel Enac

IntroductionThe incubation of airway epithelia cells at low temperatures is a common in vitro experimental approach used in the field of cystic fibrosis (CF) research to thermo-stabilise F508del-CFTR and increase its functional expression. Given that the airway epithelium includes numerous ion transporters other than CFTR, we hypothesised that there was an impact of low temperature incubation on CFTR-independent ionoregulatory mechanisms in airway epithelia derived from individuals with and without CF.MethodsAfter differentiation at the air–liquid interface, nasal epithelia were incubated at either 37°C or 29°C (low temperature) for 48 hours prior to analysis in an Ussing chamber.ResultsWhile F508del-CFTR activity was increased after low temperature incubation, activity of CFTR in non-CF epithelia was unchanged. Importantly, cultures incubated at 29°C demonstrated decreased transepithelial potential difference (TEPD) and short-circuit currents (Isc) at baseline. The predominant factor contributing to the reduced baseline TEPD and Isc in 29°C cultures was the reduced activity of the epithelial sodium channel (ENaC), evidenced by a reduced responsiveness to amiloride. This effect was observed in cells derived from both non-CF and CF donors.DiscussionSignificant transcriptional downregulation of ENaC subunits β and γ were observed, which may partially explain the decreased ENaC activity. We speculate that low temperature incubation may be a useful experimental paradigm to reduce ENaC activity in in vitro epithelial cultures.

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Gamma subunit second transmembrane domain contributes to epithelial sodium channel gating and amiloride block

AJP Renal Physiology ◽

10.1152/ajprenal.00337.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. F1585-F1592 ◽

Cited By ~ 8

Author(s):

Shujie Shi ◽

Thomas R. Kleyman

Keyword(s):

Sodium Channel ◽

Transmembrane Domain ◽

Channel Gating ◽

Epithelial Sodium Channel ◽

Open Probability ◽

Gamma Subunit ◽

Γ Subunit ◽

Inhibition Response ◽

Α And Β Subunits ◽

Pore Lining

The epithelial sodium channel (ENaC) is comprised of three homologous subunits. Channels composed solely of α- and β-subunits (αβ-channels) exhibit a very high open probability ( Po) and reduced sensitivity to amiloride, in contrast to channels composed of α- and γ-subunits or of all three subunits (i.e., αγ- and αβγ-channels). A mutant channel comprised of α- and β-subunits, and a chimeric γ-subunit where the region immediately preceding (β12 and wrist) and encompassing the second transmembrane domain (TM2) was replaced with the corresponding region of the β-subunit (γ-βTM2), displayed characteristics reminiscent of αβ-channels, including a reduced amiloride potency of block and a loss of Na+ self-inhibition (reflecting an increased Po). Substitutions at key pore-lining residues of the γ-βTM2 chimera enhanced the Na+ self-inhibition response, whereas key γ-subunit substitutions reduced the response. Furthermore, multiple sites within the TM2 domain of the γ-subunit were required to confer high amiloride potency. In summary, we have identified novel pore-lining residues of the γ-subunit of ENaC that are important for proper channel gating and its interaction with amiloride.

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Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

The Journal of General Physiology ◽

10.1085/jgp.201110763 ◽

2012 ◽

Vol 140 (4) ◽

pp. 375-389 ◽

Cited By ~ 30

Author(s):

Silke Haerteis ◽

Matteus Krappitz ◽

Alexei Diakov ◽

Annabel Krappitz ◽

Robert Rauh ◽

...

Keyword(s):

Sodium Channel ◽

Cleavage Site ◽

Double Mutant ◽

Epithelial Sodium Channel ◽

Xenopus Laevis Oocytes ◽

Cleavage Sites ◽

Channel Activation ◽

Proteolytic Activation ◽

Double Mutation ◽

Γ Subunit

Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγK189AENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γRKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γRKRK178AAAA;K189A) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.

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Stimulation of the Epithelial Sodium Channel (ENaC) by cAMP Involves Putative ERK Phosphorylation Sites in the C Termini of the Channel's β- and γ-Subunit

Journal of Biological Chemistry ◽

10.1074/jbc.m512046200 ◽

2006 ◽

Vol 281 (15) ◽

pp. 9859-9868 ◽

Cited By ~ 49

Author(s):

Li-Min Yang ◽

Ralf Rinke ◽

Christoph Korbmacher

Keyword(s):

Sodium Channel ◽

Epithelial Sodium Channel ◽

Phosphorylation Sites ◽

Γ Subunit ◽

Erk Phosphorylation ◽

Epithelial Sodium Channel Enac ◽

Stimulation Of

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The Endoplasmic Reticulum–associated Degradation of the Epithelial Sodium Channel Requires a Unique Complement of Molecular Chaperones

Molecular Biology of the Cell ◽

10.1091/mbc.e09-11-0944 ◽

2010 ◽

Vol 21 (6) ◽

pp. 1047-1058 ◽

Cited By ~ 64

Author(s):

Teresa M. Buck ◽

Alexander R. Kolb ◽

Cary R. Boyd ◽

Thomas R. Kleyman ◽

Jeffrey L. Brodsky

Keyword(s):

Sodium Channel ◽

Molecular Chaperones ◽

Essential Role ◽

Epithelial Sodium Channel ◽

Single Copy ◽

Salt Balance ◽

Expression Systems ◽

Γ Subunit ◽

Water And Salt Balance ◽

Epithelial Sodium Channel Enac

The epithelial sodium channel (ENaC) is composed of a single copy of an α-, β-, and γ-subunit and plays an essential role in water and salt balance. Because ENaC assembles inefficiently after its insertion into the ER, a substantial percentage of each subunit is targeted for ER-associated degradation (ERAD). To define how the ENaC subunits are selected for degradation, we developed novel yeast expression systems for each ENaC subunit. Data from this analysis suggested that ENaC subunits display folding defects in more than one compartment and that subunit turnover might require a unique group of factors. Consistent with this hypothesis, yeast lacking the lumenal Hsp40s, Jem1 and Scj1, exhibited defects in ENaC degradation, whereas BiP function was dispensable. We also discovered that Jem1 and Scj1 assist in ENaC ubiquitination, and overexpression of ERdj3 and ERdj4, two lumenal mammalian Hsp40s, increased the proteasome-mediated degradation of ENaC in vertebrate cells. Our data indicate that Hsp40s can act independently of Hsp70 to select substrates for ERAD.

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Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.

Journal of the American Society of Nephrology ◽

10.1681/asn.v8121813 ◽

1997 ◽

Vol 8 (12) ◽

pp. 1813-1822 ◽

Cited By ~ 11

Author(s):

A May ◽

A Puoti ◽

H P Gaeggeler ◽

J D Horisberger ◽

B C Rossier

Keyword(s):

Sodium Channel ◽

Sodium Transport ◽

Epithelial Sodium Channel ◽

Mrna Abundance ◽

Alpha Subunit ◽

Gamma Subunit ◽

Early Effect ◽

Alpha Beta ◽

Epithelial Sodium Channel Enac ◽

In Cells

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. The amiloride-sensitive epithelial sodium channel (ENaC) is a major target for the natriferic action of aldosterone. In this study, the effect of aldosterone on ENaC mRNA abundance and the rate of protein synthesis for each of the three ENaC subunits (alpha, beta and gamma) in the A6 kidney cell line were examined. In cells grown on plastic, aldosterone induced a large and rapid increase in epithelial sodium channel (ENaC) beta and gamma subunit mRNA abundance, but this effect is not translated into the synthesis of the corresponding proteins. In cells grown on a porous substrate, amiloride-sensitive electrogenic sodium transport was expressed and was upregulated by aldosterone (300 nM) as early as 1 h after the addition of the hormone. The alpha, beta, and gamma mRNA abundance was not changed by aldosterone during the first 3 h of stimulation, whereas a fourfold increase over control was observed after 24 h. The rate of synthesis of alpha subunit was significantly increased above control already 60 min after aldosterone addition, whereas beta subunit synthesis increased only 6 h after hormone addition, with no significant change for the gamma subunit. The half-lives of each subunit as assessed by 35S methionine pulse-chase experiments were short (between 40 and 50 min) and were not modified by aldosterone. Taking into account the short half-life of ENaC protein and assuming that the synthesis of the alpha subunit is a limiting factor in the assembly and expression of new channels at the cell surface, it is proposed that the aldosterone regulation of sodium transport might be, in part, mediated by de novo synthesis of the channel protein.

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