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• W2023587110 abstract "Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7–8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation. Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7–8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation. epithelial Na+ channels rat ENaC channel-activating protease Tris-buffered saline 4-morpholinepropanesulfonic acid degenerin Epithelial sodium channels (ENaC)1 play a key role in the regulation of sodium balance, extracellular fluid volume, blood pressure, and fluid reabsorption. The activity of ENaC is tightly regulated in order to ensure ion and volume homeostasis of the extra- and intracellular milieu. This regulation is under the control of several hormones and intracellular factors by mechanisms that are not yet completely understood (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1033) Google Scholar). The idea that proteases can modulate the activity of epithelial amiloride-sensitive Na+ channels is not new. Both extracellular and intracellular effects of proteases on epithelial amiloride-sensitive Na+ channels have been reported. Garty and Edelman (2Garty H. Edelman I.S. J. Gen. Physiol. 1983; 81: 785-803Crossref PubMed Scopus (112) Google Scholar) observed that extracellular trypsin, at a concentration of 1 mg/ml, induced an irreversible inhibition of the sodium transport in toad urinary bladder and that this effect could be prevented by amiloride. Lewis and Alles (3Lewis S.A. Alles W.P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5345-5348Crossref PubMed Scopus (43) Google Scholar) studied the effects of proteases such as kallikrein, urokinase, and plasmin and observed that these proteases converted a normally highly selective amiloride-sensitive Na+ channel into a nonselective cation channel. Chraibi et al. (4Chraibi A. Vallet V. Firsov D. Hess S.K. Horisberger J.D. J. Gen. Physiol. 1998; 111: 127-138Crossref PubMed Scopus (168) Google Scholar) found that trypsin, at a concentration of 2 μg/ml, increased the amiloride-sensitive current up to 20-fold when added to the bathing solution of ENaC-expressingXenopus oocytes. Jovov et al. (5Jovov B. Wills N.K. Donaldson P.J. Lewis S.A. Am. J. Physiol. 1990; 259: C869-C882Crossref PubMed Google Scholar) demonstrated that A6 cells (derived from Xenopus laevis kidney) express and secrete a kallikrein-like serine protease from their apical side. This serine protease has been cloned and named channel-activating protease (CAP1) (6Vallet V. Chraibi A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (446) Google Scholar). Masilamani et al. (7Masilamani S. Kim G.H. Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (621) Google Scholar) suggested that activation of Na+ currents by aldosterone is mediated by CAP1 that in turn cleaves γ-ENaC in the early portion of extracellular loop (7Masilamani S. Kim G.H. Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (621) Google Scholar). Until now, this hypothesis has not been tested. An intracellular effect of trypsin on ENaC immunopurified from human lymphocytes and incorporated in planar lipid bilayers was reported by Ismailov et al. (8Ismailov I.I. Berdiev B.K. Fuller C.M. Bradford A.L. Lifton R.P. Warnock D.G. Bubien J.K. Benos D.J. Am. J. Physiol. 1996; 270: C214-C223Crossref PubMed Google Scholar) who studied Liddle's disease. Liddle's disease is a form of human hypertension caused by mutations in the β- and γ-subunit of ENaC. Many of the described mutations result in truncation of the C termini of either the β- or γ-subunit of ENaC. It is likely that the increased channel activity seen in patients with Liddle's disease is due to both increased ENaC cell surface expression and increased channel open probability (9Ji H.L. Fuller C.M. Benos D.J. J. Biol. Chem. 1999; 274: 37693-37704Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Ismailov et al.(8Ismailov I.I. Berdiev B.K. Fuller C.M. Bradford A.L. Lifton R.P. Warnock D.G. Bubien J.K. Benos D.J. Am. J. Physiol. 1996; 270: C214-C223Crossref PubMed Google Scholar) demonstrated that the addition of trypsin (0.5 mg/ml) to the putative intracellular side of lymphocyte Na+ channels incorporated into planar lipid bilayers increased the single channel open probability (Po) in normal individuals as compared with that seen in lymphocyte Na+ channels obtained from patients with Liddle's disease. Intracellular addition of trypsin to lymphocyte Na+ channels obtained from patients with Liddle's disease did not further increase single channelPo.Recent analysis of protein motifs in some members of the DEG/ENaC family revealed the presence of protease prodomains and protease inhibitor motifs. The presence of a Kunitz-type protease inhibitor motif that inhibits serine proteases was described by Tavernarakis et al. (10Tavernarakis N. Everett J.K. Kyrpides N.C. Driscoll M. Curr. Biol. 2001; 11: R205-R208Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) in the N terminus of α-ENaC subunit. In addition, two nematode proteins, MEC-2 and UNC-1, contain a domain implicated in proteolysis of membrane-associated proteins (11Tavernarakis N. Driscoll M. Kyrpides N.C. Trends Biochem. Sci. 1999; 24: 425-427Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). These finding led to the suggestion that members of the DEG/ENaC family may be regulated by, or may themselves participate in, proteolysis as an important part of the regulatory mode of these channels (11Tavernarakis N. Driscoll M. Kyrpides N.C. Trends Biochem. Sci. 1999; 24: 425-427Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar).Despite numerous biophysical studies that demonstrated the functional effects of serine proteases on ENaC, there are no biochemical data demonstrating proteolysis of any ENaC subunit by trypsin. In this study, we showed that in vitro translated β- and γ-subunits of ENaC can be cleaved by trypsin in the nanogram/ml range and that the most susceptible trypsin cleavage sites are in the C termini of these β- and γ-subunits. We also demonstrate that 2 μg/ml trypsin (the lowest concentration used in functional studies so far) is enough to digest completely each of the three ENaC subunits.RESULTSWe have shown previously that exposure of the “cytoplasmic” surface of purified lymphocyte Na+ channel protein incorporated into planar bilayers to 0.5 mg/ml trypsin increasedPo 5-fold. Moreover, these trypsinized channels resembled, in their conductance and gating characteristics, channels purified from lymphocytes obtained from confirmed Liddle's patients (8Ismailov I.I. Berdiev B.K. Fuller C.M. Bradford A.L. Lifton R.P. Warnock D.G. Bubien J.K. Benos D.J. Am. J. Physiol. 1996; 270: C214-C223Crossref PubMed Google Scholar). To test whether trypsin produced comparable effects on ENaC, we transiently transfected 3T3-fibroblasts with α-, β-, γ-rENaC and recorded ENaC in excised patches using the inside-out mode of the patch clamp technique. As can be seen in Fig.1, 5-pS, Na+-selective channels were recorded. After a 5-min exposure to 0.25 mg/ml trypsin, single channel Po increased, and the channel remained essentially in its open state. No effect was seen of 50 μg/ml intracellular trypsin after 5 min of exposure onPo recorded from inside-out patches of ENaC-expressing oocyte membranes (not shown). These results are comparable with those reported for the lymphocyte channel (8Ismailov I.I. Berdiev B.K. Fuller C.M. Bradford A.L. Lifton R.P. Warnock D.G. Bubien J.K. Benos D.J. Am. J. Physiol. 1996; 270: C214-C223Crossref PubMed Google Scholar). Because CAPs are thought to work from the extracellular surface of the membrane (6Vallet V. Chraibi A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (446) Google Scholar), we tested the effects of extracellular trypsin on ENaC activity. For these experiments we used ENaC incorporated into planar lipid bilayers (Fig. 2). The addition of 0.25 mg/ml trypsin to the trans (i.e. outside) bathing solution activated ENaC. The lowest concentration of trypsin that can affect ENaC incorporated into planar lipid bilayers from either side was 200 μg/ml. However, this activation was time-dependent. Prolonged exposure resulted in a deterioration of the functional integrity of the channel and, ultimately, loss of any discernible gating. Thus, both extracellular and intracellular trypsin can “activate” ENaC.Figure 2Effect of trans(extracellular) trypsin on ENaC reconstituted into planar lipid bilayers. Bilayers were bathed with symmetrical 100 mmNaCl, 10 mm MOPS. Trypsin was added to transcompartment of the bilayer chamber in the concentration indicated. Holding potential was +100 mV and referred to the virtually groundedtrans chamber. For illustration purposes records shown were digitally filtered at 100 Hz using pCLAMP software (Axon Instruments). Corresponding all-point amplitude histograms were constructed by pCLAMP from a record of at least 5 min in length.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Masilamani et al. (7Masilamani S. Kim G.H. Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (621) Google Scholar) hypothesized that CAP1 activates ENaC by a proteolytic cleavage mechanism in the first portion of the extracellular loop of the γ-ENaC subunit. To test this hypothesis, we prepared a construct of γ-ENaC truncated at a predicted proteolytic site (Δ2–136) and examined amiloride-sensitive Na+currents in oocytes expressing wild type α-, β-, γ-rENaC, or rENaC that contained this N-terminal truncated (Δ2–138) γ-rENaC subunit (Fig. 3). Amiloride-sensitive Na+ currents in oocytes expressing this construct were significantly lower than in oocytes expressing wild type rENaC. This finding is in contrast to the predicted activation of ENaC by CAP1 proteolytic cleavage of the γ-subunit. These findings were expected because we have found previously (17Chalfant M.L. Denton J.S. Langloh A.L. Karlson K.H. Loffing J. Benos D.J. Stanton B.A. J. Biol. Chem. 1999; 274: 32889-32896Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) that elimination of the N terminus of the γ-subunit diminishes macroscopic Na+currents in oocytes. As further support of this idea, experiments were conducted in planar lipid bilayers into which wild type α, wild type β, and N-terminal truncated (Δ2–138) γ-rENaC were incorporated. The single channels that were seen were indistinguishable from the wild type, i.e. no change in single channel conductance or open probability was measured, suggesting that the elimination of the early portion of the γ-subunit was not detrimental to the integrity of the functional channels recorded (data not shown). Although the mechanisms by which serine proteases activate ENaC are not clear, functional studies demonstrated that both extracellular and intracellular trypsin can activate ENaC.Because all three rENaC subunits contain numerous trypsin proteolytic sites, our goal in these biochemical studies was to discover the most susceptible trypsin proteolytic cleavage sites within the rENaC subunits. Immunopurified, epitope tag-labeled rENaC subunits (synthesized by in vitro transcription and translation) were used as a source of subunit proteins for trypsin digestion. A schematic representation of each ENaC subunit with corresponding truncation points and positions of epitope tags in each subunit is shown in Fig.4. The locations of the N- and C-terminal truncations of β- and γ-rENaC subunits are labeled bydouble horizontal lines, and the number of the last amino acid before the truncation is indicated. The position of the epitope tag in the extracellular loop of each subunit is shown as asmall black box with the number of the starting and ending amino acid. We used non-glycosylated forms of in vitrotranslated proteins (the transcription mixture did not contain microsomes) for simplicity. Transcription of each construct in this condition resulted in a protein band of the appropriate size (Figs.Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, lane 1). Trypsinization was performed in TBS at 37 °C for 2 h. By using different concentrations of trypsin, we determined that 2 μg/ml trypsin was sufficient to digest completely all three subunits of ENaC (Figs. 5, A andB, 6, A and B, and 8A). To unmask the most susceptible proteolytic site in each subunit, we decreased the concentration of trypsin 1000-fold and exposed each protein subunit to four different concentrations of trypsin, namely 0.002, 0.02, 0.2, and 2 μg/ml. The proteolytic mixture was analyzed using both 8 and 15% SDS-PAGE. Proteolysis of the α-subunit by trypsin in the range of 0.002–0.2 μg/ml did not result in any visible proteolytic fragments (Fig. 5,A and B). However, 2 μg/ml trypsin completely disintegrated the α-rENaC subunit (Fig. 5, A andB). We also examined the time course of proteolysis by trypsin at 5, 10, 20, and 40 min using 0.02 μg/ml trypsin (data not shown). We did not observe any visible proteolytic fragments at these time points. These results suggest that either all α-rENaC trypsinization sites have a similar susceptibility to trypsin or our experimental conditions are not sensitive enough to detect subtle differences in trypsin effects. Tavarnarakis et al. (10Tavernarakis N. Everett J.K. Kyrpides N.C. Driscoll M. Curr. Biol. 2001; 11: R205-R208Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar) described the presence of a Kunitz-type protease inhibitor motif in the N terminus of α-ENaC subunit. It is possible that this protease inhibitor motif protects the α-rENaC subunit from proteolysis at low trypsin concentrations, but 2 μg/ml trypsin is sufficient to overcome this inhibitory effect. By using the same experimental conditions and detection techniques, we found that trypsinization of β-rENaC with 0.002–0.2 μg/ml trypsin resulted in proteolytic fragments (Fig.6A and Fig. 5B). Analysis using 8% SDS-gel electrophoresis (Fig. 6A) revealed that trypsinization of β-rENaC produced a double band. The higher band had the same molecular mass as the non-cut protein, and the lower band was 7–8 kDa lower than the non-cut band protein, suggesting a reduction in size due to trypsinization. Similar results were obtained by trypsinization of β-rENaC with 0.02 μg/ml trypsin at 5-, 10-, 20-, and 40-min time points (data not shown). All samples measured at each of these time points contained double bands. There was a decrease in the intensity of the higher band with a concomitant increased intensity of the lower band with time, consistent with a time-dependent proteolytic process. Analysis of trypsinized β-rENaC using 15% SDS-gel electrophoresis (Fig. 6B) revealed the presence of a small (7–8 kDa) band that was visible when 0.02 or 0.2 μg/ml was used for trypsinization. Detection of this 7–8-kDa band on a 15% gel suggests that the reduction in size of β-rENaC resulted from proteolytic cleavage at one side of the protein chain, rather than occurring at multiple sites.Figure 4Transmembrane model of epitope tag α-, β-, and γ-rENaC subunits, including points of N- and C-terminal truncation of β- and γ-rENaC used in this study. Black numbers correspond to amino acids bordering the transmembrane segments. The location of the short peptides used to produce antibodies is represented by small black boxes (Tag Ab, 272, 260; Tag Ab, 171, 159, and Flag Ab, 149, 143) in the extracellular loop. Corresponding numbers indicate the starting and ending amino acids of the inserted tag peptide. Position of the truncation (trc) in C or N termini of β- and γ-rENaC are labeled bydouble black lines, with the number indicating the last amino acid before truncation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Trypsinization of immunopurified α-rENaC. α-rENaC was transcribed and translated in vitro (IN V) using the TNT transcription/translation system (Promega) and subjected to immunoprecipitation in RIPA buffer. Immunopurified protein was then exposed to trypsin (Tryp) at concentrations ranging from 0.002 to 2 μg/ml in TBS buffer for 2 h and analyzed using SDS-PAGE. A, analysis of proteolytic fragments using 8% SDS-PAGE. B, analysis of proteolytic fragments using 15% SDS-PAGE. Proteolysis of the α-subunit by trypsin in the range of 0.002 to 0.2 μg/ml did not result in any visible proteolytic fragments. However, 2 μg/ml trypsin completely digested the α-ENaC subunit.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Trypsinization of immunopurified β-rENaC. β-rENaC was transcribed and translated in vitro using TNT transcription/translation system (Promega) and subjected to immunoprecipitation (IP) in RIPA buffer. Immunopurified protein was then exposed to trypsin (Tryp) in concentrations ranging from 0.002 to 2 μg/ml in TBS buffer for 2 h and analyzed using SDS-PAGE. A, analysis of proteolytic segments using 8% SDS-PAGE. The trypsinization of β-rENaC with 0.002–0.2 μg/ml trypsin resulted in a double band. The higher band had the same molecular mass as the non-cut protein, and the lower band was 7–8 kDa lower than the non-cut band protein, suggesting a reduction in size due to trypsinization. B, analysis of proteolytic segments using 15% SDS-PAGE. The small (7–8 kDa) band is visible in lanes where 0.02 or 0.2 μg/ml was used for trypsinization. Detection of this 7–8-kDa band on the 15% gel suggests that the reduction in size of β-rENaC is a result of proteolytic cleavage at one side of the protein chain, as opposed to multiple cleavage sites.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Trypsinization of immunopurified γ-rENaC. γ-rENaC was transcribed and translated in vitro (IN V) using TNT transcription/translation system (Promega) and subjected to immunoprecipitation (IP) in RIPA buffer. Immunopurified protein was then exposed to trypsin in concentrations ranging from 0.002 to 2 μg/ml in TBS buffer for 2 h and analyzed using SDS-PAGE. A, analysis of proteolytic segments using 8% SDS-PAGE. After trypsinization using 0.002–0.2 μg/ml trypsin (Tryp) γ-rENaC protein ran as a double band. The higher band ran at ∼71 kDa (same as non-trypsinized protein) and the lower band ran at 63 kDa (7–8 kDa difference in molecular mass), suggesting a reduction in size due to trypsinization. B, analysis of proteolytic segments using 15% SDS-PAGE. The presence of a 7–8-kDa band on a 15% gel after trypsinization with 0.02–0.2 μg/ml suggests that the reduction in size of γ-rENaC is a result of proteolytic cleavage at one proteolytic site, as opposed to multiple cleavage sites.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Trypsinization of immunopurified C- or N-terminally truncated β-rENaC. C- or N-terminally truncated β-rENaC was transcribed and translatedin vitro (IN V) using TNT transcription/translation system (Promega) and subjected to immunoprecipitation in RIPA buffer. Immunopurified protein was then exposed to trypsin (Tryp) in concentrations ranging from 0.002 to 2 μg/ml in TBS buffer for 2 h and analyzed using 8% SDS-PAGE. A, trypsinization of N-terminally truncated β-rENaC. Trypsinization of N-terminally truncated β-rENaC using 0.002–0.2 μg/ml resulted in the appearance of a double band, a higher band with the same molecular mass as the non-trypsinized protein, and a lower band that is 7–8 kDa lower than the non-trypsinized protein. B, trypsinization of C-terminally truncated β-rENaC. Trypsinization of C-terminally truncated β-rENaC using trypsin in the range from 0.002 to 0.2 μg/ml did not reveal the presence of any proteolytic fragments, just a single band without any shift in molecular mass. C, amino acid sequence of β-rENaC in close proximity (±7 amino acids) to C-terminal truncation (574). Amino acids in italics are the predicted trypsin proteolytic sites.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Trypsinization of immunopurified C- or N-terminally truncated γ-rENaC. C- or N-terminally truncated γ-rENaC was transcribed and translatedin vitro (IN V) using TNT transcription/translation system (Promega) and subjected to immunoprecipitation in RIPA buffer. Immunopurified proteins were exposed to trypsin (Tryp) in concentrations ranging from 0.002 to 2 μg/ml in TBS buffer for 2 h and analyzed using 8% SDS-PAGE. A, trypsinization of N-terminally truncated γ-rENaC. Trypsinization of N-terminally truncated γ-rENaC using 0.002–0.2 μg/ml resulted in the appearance of a double band. The higher band was at the same molecular mass as the non-trypsinized protein, and the lower band was 7–8 kDa smaller than non-trypsinized γ-rENaC. B, trypsinization of C-terminally truncated γ-rENaC. Trypsinization of C-terminally truncated γ-rENaC, using trypsin in ranges from 0.002 to 0.2 μg/ml, did not reveal any proteolytic fragments. Only one band without a shift in molecular mass was observed. C, amino acid sequence of γ-rENaC in close proximity (±7 amino acids) to C-terminal truncation (574). Amino acids in italics are the predicted trypsin proteolytic sites.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To address further the question from which side (C or N terminus) trypsin cleaves β-rENaC, we used C- and N-terminally truncated constructs of β-rENaC (see Fig. 4). The N-terminally truncated construct had a deletion of 49 amino acids at the N terminus, whereas the C-terminally truncated construct had a deletion of the last 74 amino acids from the C terminus. Our hypothesis was that if trypsin cuts the C terminus of β-rENaC in close proximity to the point of truncation, the C-terminally truncated construct would be unaffected. In this case, the N-terminally truncated construct would be cut, resulting in a similar pattern as non-truncated β-rENaC (just lower molecular mass) and vice versa. The results of these experiments are shown in Fig. 7. Trypsinization of N-truncated β-rENaC using 0.002–0.2 μg (Fig. 7A) resulted in the appearance of a doublet on 8% gels, a higher band with the same molecular mass as the non-trypsinized protein, and band that was 7–8 kDa lower than the non-trypsinized protein. Trypsinization of C-terminally truncated β-rENaC using trypsin in the range from 0.002 to 0.2 μg/ml did not reveal any proteolytic fragments; a single band without any shift in molecular mass was observed after trypsin treatment (Fig. 9B). These results suggest that the site of trypsin cleavage occurs within the C terminus domain of β-rENaC. Indeed, analysis of the protein structure of β-rENaC in close proximity (±7 amino acids) from the point of the C-terminal truncation (amino acid 564) revealed the presence of five potential trypsin cleavage sites, i.e. four arginines and one lysine (Fig.7C). Thus, we suggest that the most susceptible trypsin cleavage site within β-rENaC is in close proximity to the point of truncation at amino acid 564 of the C-terminally truncated β-rENaC used in this study. This C-terminally truncated β-rENaC is identical to the β-ENaC truncation mutant (R564stop) identified in the proband of Liddle's disease (18Tamura H. Schild L. Enomoto N. Matsui N. Marumo F. Rossier B.C. J. Clin. Invest. 1996; 97: 1780-1784Crossref PubMed Scopus (249) Google Scholar).We next tested the effect of trypsinization on the γ-rENaC subunit (Fig. 8, A and B). Analysis of the proteolytic mixture using an 8% SDS gel (Fig.6A) revealed that γ-rENaC ran as a doublet following trypsinization (0.002–0.2 μg). The higher band ran at ∼71 kDa (the same as the non-trypsinized protein), and the lower band ran at 63 kDa (a 7–8 kDa difference in molecular mass) suggesting a reduction in size due to trypsinization. The presence of this 7–8-kDa band on a 15% gel (Fig. 8B) subsequent to trypsinization suggested that the reduction in size of γ-rENaC was a result of proteolytic cleavage at one proteolytic site, as opposed to multiple cleavage sites. The appearance of a doublet in 8% gels was also seen following trypsinization of γ-rENaC with 0.02 μg/ml trypsin at time points of 5, 10, 20, and 40 min (data not shown). A double band with decreasing intensity of the higher molecular mass band and increasing intensity of the lower band was observed as a function of time. To delineate further whether the C or N terminus of γ-rENaC was cleaved by trypsin, we used C- and N-terminally truncated constructs of γ-rENaC (Fig. 4). The N-terminally truncated γ-rENaC construct was missing the first 53 amino acids. The C-terminally truncated construct had the last 75 amino acids deleted. Our hypothesis was the same as that for β-rENaC, namely that if trypsin cut the C terminus of γ-rENaC, the C-terminally truncated construct will not be cut and the N-terminally truncated construct will be cut. The results of these experiments are shown in Fig. 8. Incubation of N-truncated γ-rENaC with 0.002–0.2 μg of trypsin (Fig. 8A) resulted in the appearance of a doublet on an 8% gel. The higher band had the same molecular mass as the non-trypsinized protein, and the lower band was 7–8 kDa smaller than the non-trypsinized protein. This result suggested that trypsin cut the C terminus of γ-rENaC in close proximity to amino acid 574. Again, an analysis of the protein structure of the C terminus of γ-rENaC revealed the presence of four potential trypsin cleavage sites, namely two lysine and two arginines (Fig.9C). Trypsinization of C-terminally truncated γ-rENaC did not reveal any proteolytic fragments. Only one band without a shift in molecular weight was observed (Fig. 9B). This result supports our finding that the most susceptible trypsin site of γ-rENaC is in very close proximity to amino acid 574. This C-terminally truncated γ-rENaC is identical to the truncation mutant (R574stop) identified in Liddle's disease (18Tamura H. Schild L. Enomoto N. Matsui N. Marumo F. Rossier B.C. J. Clin. Invest. 1996; 97: 1780-1784Crossref PubMed Scopus (249) Google Scholar).DISCUSSIONOur functional studies confirmed findings from other laboratories (4Chraibi A. Vallet V. Firsov D. Hess S.K. Horisberger J.D. J. Gen. Physiol. 1998; 111: 127-138Crossref PubMed Scopus (168) Google Scholar, 6Vallet V. Chraibi A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. Nature. 1997; 389: 607-610Crossref PubMed Scopus (446) Google Scholar, 8Ismailov I.I. Berdiev B.K. F" @default.
• W2023587110 created "2016-06-24" @default.
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• W2023587110 date "2002-02-01" @default.
• W2023587110 modified "2023-10-16" @default.
• W2023587110 title "The Serine Protease Trypsin Cleaves C Termini of β- and γ-Subunits of Epithelial Na+ Channels" @default.
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• W2023587110 doi "https://doi.org/10.1074/jbc.m108354200" @default.
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