Haerteis S et al. (2012), Proteolytic activation of the epithelial sodium...

XB-ART-45727

Pflugers Arch 2012 Oct 01;4644:353-65. doi: 10.1007/s00424-012-1138-3.

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Proteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S.

Haerteis S , Krappitz M , Bertog M , Krappitz A , Baraznenok V , Henderson I , Lindström E , Murphy JE , Bunnett NW , Korbmacher C .

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Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.

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Species referenced: Xenopus laevis
Genes referenced: cat.2 cela1.2 cela1.6 cfd prss1 prtn3

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	Fig. 1. Cat-S stimulates ENaC currents in Xenopus laevis oocytes expressing human ENaC. aâd Oocytes expressing human ENaC were incubated for 30Â min in protease-free solution (control) or in a solution containing either Cat-S (1Â Î¼M) or chymotrypsin (2Â Î¼g/ml). Amiloride-sensitive whole-cell currents (ÎIami) were determined before (â) and after (+) incubation. Six representative whole-cell current traces from one batch of oocytes are shown. aâc Amiloride (ami) was present in the bath solution to specifically inhibit ENaC as indicated by black bars. d Individual ÎIami values from a representative experiment using one batch of oocytes. Data points obtained from an individual oocyte are connected by a line
	Fig. 2. Stimulatory effect of Cat-S on human ENaC is concentration dependent. Oocytes expressing human Î±Î²Î³ ENaC were incubated for 30Â min in protease-free solution (control), in solutions containing different concentrations of Cat-S (0.01, 0.03, 0.1, 0.3, 1, and 3Â Î¼M) or in a solution containing chymotrypsin (2Â Î¼g/ml). Amiloride-sensitive whole-cell currents (ÎIami) were detected before (ÎIami initial) and after incubation (ÎIami 30Â min). Columns represent the relative stimulatory effect on ÎIami calculated as the ratio of ÎIami 30Â min/ÎIami initial. Numbers inside the columns indicate the number of individual oocytes measured. N indicates the number of different batches of oocytes
	Fig. 3. Activation of ENaC by Cat-S is prevented by the Cat-S inhibitor LHVS. Oocytes expressing human ENaC were incubated for 30Â min in protease-free solution (control), in chymotrypsin (2Â Î¼g/ml), in Cat-S (1Â Î¼M), in LHVS (2Â Î¼M), or in a solution containing a combination of Cat-S (1Â Î¼M) and LHVS (5Â Î¼M). Amiloride-sensitive whole-cell currents (ÎIami) were determined before (â) and after (+) incubation. The bar diagram represents normalized average results obtained in five different batches of oocytes (Nâ=â5). The individual ÎIami values were normalized to the mean ÎIami value of the ENaC-expressing control group in protease-free solution. Numbers inside the columns indicate the number of individual oocytes measured. pâ<â0.01, *pâ<â0.001, paired t test
	Fig. 4. The Cat-S inhibitor LHVS has no effect on ENaC activation by the serine proteases trypsin and chymotrypsin. a Oocytes expressing human ENaC were incubated for 30Â min in protease-free solution (control), in solutions with different concentrations of chymotrypsin (0.02, 0.2, and 2Â Î¼g/ml), in LHVS (2Â Î¼M), or in solutions containing a combination of different concentrations of chymotrypsin (0.02, 0.2, and 2Â Î¼g/ml) and LHVS (2Â Î¼M). Amiloride-sensitive whole-cell currents (ÎIami) were determined before (â) and after (+) incubation. Columns represent relative stimulatory effect on ÎIami calculated as the ratio of ÎIami measured after 30Â min of incubation (ÎIami 30Â min) to the initial ÎIami (ÎIami initial) measured before incubation. Numbers inside the columns indicate the number of individual oocytes measured. b Similar experiment as shown in a using the serine protease trypsin (2Â Î¼g/ml) instead of chymotrypsin
	Fig. 5. Cat-S can activate ENaC in an acidic environment. Oocytes expressing human ENaC were incubated for 30Â min in protease-free (control), in chymotrypsin (0.2Â Î¼g/ml), or in Cat-S (1Â Î¼M) solution with a physiological pH of 7.4 (white columns) or an acidic pH of 5 (black columns). Amiloride-sensitive whole-cell currents (ÎIami) were determined before (â) and after (+) incubation. a ÎIami values from a representative experiment using one batch of oocytes. b Summary of similar experiments as shown in a. Columns represent relative stimulatory effect on ÎIami calculated as the ratio of ÎIami measured after 30Â min of incubation (ÎIami 30Â min) to the initial ÎIami (ÎIami initial) measured before incubation. Numbers inside the columns indicate the number of individual oocytes measured. N indicates the number of different batches of oocytes. pâ<â0.05, **pâ<â0.001, unpaired t test
	Fig. 6. In vitro cleavage analysis of the 23-mer Î³ENaC peptide suggests that Cat-S may cleave human Î³ENaC at more than one cleavage site. a Sequence of the 23-mer Î³ENaC peptide showing putative cleavage sites for proteolytic ENaC activation. b The 23-mer Î³ENaC peptide (500Â Î¼M) was incubated with Cat-S (1Â Î¼M) for 30Â min and cleavage products were identified by HPLC and mass spectrometry. Cat-S degraded the 23-mer Î³ENaC peptide showing four products detected by HPLC
	Fig. 7. Activation of ENaC by Cat-S generates a Î³ENaC cleavage product at the cell surface indicating proteolytic channel activation. The Cat-S inhibitor LHVS prevented the generation of an additional cleavage product at the cell surface. Oocytes expressing human ENaC were incubated for 30Â min in protease-free solution (control), in chymotrypsin (2Â Î¼g/ml), in Cat-S (1Â Î¼M), or in a solution containing a combination of Cat-S (1Â Î¼M) and LHVS (5Â Î¼M). Expression of biotinylated Î³ENaC at the cell surface was analyzed by SDS-PAGE. Î³ENaC was detected with an antibody against the C-terminus of human Î³ENaC. In non-injected (ni) oocytes, Î³ENaC-specific signals were absent. âââ indicates an empty lane on the gel. Molecular weight markers are shown on the left side of the gel. Representative western blot from one batch of oocytes
	Fig. 8. Mutating two putative neutrophil elastase cleavage sites (Î³V182;V193) prevents proteolytic activation of Î³ENaC by Cat-S. Oocytes expressing Î±Î²Î³ (open symbols) or Î±Î²Î³V182G;V193GENaC (filled symbols) were incubated for 30Â min in protease-free solution (control) or in a solution containing either hNE (10Â Î¼g/ml) or Cat-S (1Â Î¼M). Amiloride-sensitive whole-cell currents (Î”Iami) were determined before (âˆ’) and after (+) incubation. a Individual Î”Iami values from a representative experiment using one batch of oocytes. Data points obtained from individual oocytes are connected by a line. b Summary of similar experiments as shown in a. Columns represent relative stimulatory effect on Î”Iami calculated as the ratio of Î”Iami measured after 30Â min of incubation (Î”Iami 30Â min) to the initial Î”Iami (Î”Iami initial) measured before incubation. Numbers inside the columns indicate the number of individual oocytes measured. N indicates the number of different batches of oocytes
	Fig. 9. Cat-S stimulates ENaC in confluent M-1 mouse collecting duct cells. a Representative equivalent short-circuit current (ISC) recordings from confluent M-1 cells pretreated with nafamostate mesylate to reduce constitutive ENaC activation by endogenous proteases. Vehicle control (phosphate buffered saline, upper trace) or Cat-S (2Â Î¼M, lower trace) was added to the apical bath solution of M-1 cells. At the end of the experiment, amiloride (ami; 10Â Î¼M) was added apically to confirm that the stimulated ISC was mediated by ENaC. b, c Summary of results from similar experiments as shown in a. bData points represent individual ISC values obtained from six (control) or seven (Cat-S) individual experiments that are connected by a line. cColumns represent relative stimulatory effect on ÎISC calculated as the ratio of ÎISC measured after 20Â min of incubation (ÎISC 20Â min) to the initial ISC (ÎISC initial) measured before apical addition. **pâ<â0.01, unpaired t test

References [+] :

Adebamiro, A segment of gamma ENaC mediates elastase activation of Na+ transport. 2007, Pubmed