TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ∼70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ∼70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ∼70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.

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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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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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SARS-CoV-2 Origin: An Affair of Codons?

10.20944/preprints202106.0121.v1 ◽

2021 ◽

Author(s):

Antonio Ramón Romeu ◽

Enric Ollé

Keyword(s):

Human Genome ◽

Genetic Markers ◽

Cleavage Site ◽

Cleavage Sites ◽

Furin Cleavage ◽

Large Sample ◽

The Core ◽

Furin Cleavage Site

The furin cleavage site, with an arginine doublet (RR), is one of the clues of the SARS-CoV-2 origin. This furin-RR is encoded by the CGG-CGG sequence. Because arginine can be encoded by six codons, in a previous work we found that in SARS-CoV-2, CGG was the minority arginine codon (3%). Also, analyzing the RR doublet from a large sample of furin cleavage sites of several kinds of viruses, we found that none of them were encoded by CGG-CGG. Here, we come back to the core of the matter, but from the perspective that in the human genome, in contrast, CGG is the majoroty arginine codon (21%). Here, we highlighted that the 6 arginine codons provide genetic markers to a traceability on the RR origin in the furin site, as well as, to weigh the probability of the theories about the origin of the virus.

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Experimental nephrotic syndrome leads to proteolytic activation of the epithelial sodium channel (ENaC) in the mouse kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00199.2021 ◽

2021 ◽

Author(s):

Bernhard N. Bohnert ◽

Daniel Essigke ◽

Andrea Janessa ◽

Jonas C Schneider ◽

Matthias Wörn ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Sodium Channel ◽

Serine Protease ◽

Cleavage Site ◽

Mineralocorticoid Receptor ◽

Epithelial Sodium Channel ◽

Sodium Retention ◽

Cleavage Sites ◽

Proteolytic Activation ◽

Epithelial Sodium Channel Enac

Proteolytic activation of the renal epithelial sodium channel ENaC involves cleavage events in its α- and γ-subunits and is thought to mediate sodium retention in nephrotic syndrome (NS). However, detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α- and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the N-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed sodium retention and increased expression of fragments of α- and γ-ENaC cleaved at both the proximal and more prominently at the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented sodium retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α- and γ-ENaC was similarly found in healthy mice treated with a low salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and more prominently distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.

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A Novel Pathway of Epithelial Sodium Channel Activation Involves a Serum- and Glucocorticoid-inducible Kinase Consensus Motif in the C Terminus of the Channel's α-Subunit

Journal of Biological Chemistry ◽

10.1074/jbc.m403260200 ◽

2004 ◽

Vol 279 (37) ◽

pp. 38134-38142 ◽

Cited By ~ 133

Author(s):

Alexei Diakov ◽

Christoph Korbmacher

Keyword(s):

Sodium Channel ◽

Epithelial Sodium Channel ◽

Channel Activation ◽

Α Subunit ◽

Consensus Motif ◽

C Terminus ◽

Sodium Channel Activation

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SARS-CoV-2 and MERS-CoV Share the Furin Site CGG-CGG Genetic Footprint

10.20944/preprints202110.0080.v2 ◽

2021 ◽

Author(s):

Antonio R. Romeu

Keyword(s):

Middle East ◽

Markov Model ◽

Cleavage Site ◽

Spike Protein ◽

Cleavage Sites ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Arginine Residues ◽

Codon Pair ◽

Shed Light

The SARS-CoV-2 polybasic furin cleavage site is still a missing link. Remarkably, the two arginine residues of this protease recognition site are encoded by the CGG codon, which is rare in Betacoronavirus. However, the arginine pair is common at viral furin cleavage sites, but are not CGG-CGG encoded. The question is: Is this genetic footprint unique to the SARS-CoV-2? To address the issue, using Perl scripts, here I dissect in detail the NCBI Virus database in order to report the arginine dimers of the Betacoronavirus proteins. The main result reveals that a group of Middle East respiratory syndrome-related coronavirus (MERS-CoV) (isolates: camel/Nigeria/NVx/2016, host: Camelus dromedarius) also have the CGG-CGG arginine pair in the spike protein polybasic furin cleavage region. In addition, CGG-CGG encoded arginine pairs were found in the orf1ab polyprotein from HKU9 and HKU14 Betacoronavirus, as well as, in the nucleocapsid phosphoprotein from few SARS-CoV-2 isolates. To quantify the probability of finding the arginine CGG-CGG codon pair in Betacoronavirus, the likelihood ratio (LR) and a Markov model were defined. In conclusion, it is highly unlikely to find this genetic marker in betacoronaviruses wildlife, but they are there. Collectively, results shed light on recombination as origin of the virus CGG-CGG arginine pair in the S1/S2 cleavage site.

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Global Diversification and Distribution of Coronaviruses With Furin Cleavage Sites

Frontiers in Microbiology ◽

10.3389/fmicb.2021.649314 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiaotong Liu ◽

Qunfu Wu ◽

Zhigang Zhang

Keyword(s):

Cleavage Site ◽

Evolutionary History ◽

Plasma Membranes ◽

Cleavage Sites ◽

Furin Cleavage ◽

Furin Cleavage Site ◽

Global Epidemiology ◽

History Of ◽

Animal Hosts ◽

Hydrolysis Of

Knowledge about coronaviruses (CoVs) with furin cleavage sites is extremely limited, although these sites mediate the hydrolysis of glycoproteins in plasma membranes required for MERS-CoV or SARS-CoV-2 to enter cells and infect humans. Thus, we have examined the global epidemiology and evolutionary history of SARS-CoV-2 and 248 other CoVs with 86 diversified furin cleavage sites that have been detected in 24 animal hosts in 28 countries since 1954. Besides MERS-CoV and SARS-CoV-2, two of five other CoVs known to infect humans (HCoV-OC43 and HCoV-HKU1) also have furin cleavage sites. In addition, human enteric coronavirus (HECV-4408) has a furin cleavage site and has been detected in humans (first in Germany in 1988), probably via spillover events from bovine sources. In conclusion, the presence of furin cleavage sites might explain the polytropic nature of SARS-CoV-2- and SARS-CoV-2-like CoVs, which would be helpful for ending the COVID-19 pandemic and preventing outbreaks of novel CoVs.

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