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ENaC proteolytic regulation by channel-activating protease 2.

García-Caballero A, Dang Y, He H, Stutts MJ - J. Gen. Physiol. (2008)

Bottom Line: Potential therapies for disorders of Na(+) absorption require better understanding of ENaC regulation.Replacement of gamma-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I(Na) and the 75-kD gamma-ENaC fragment.These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in gamma-ENaC.

View Article: PubMed Central - PubMed

Affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599, USA. acaballe@med.unc.edu

ABSTRACT
Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na(+) absorption across epithelial surfaces throughout the body. Excessive Na(+) absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na(+) absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (alpha-K561, beta-R503, and gamma-R515). Sites in alpha-ENaC at K149/R164/K169/R177 and furin-consensus sites in alpha-ENaC (R205/R231) and gamma-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in gamma-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (alpha,beta,gammaR138A/Q) completely abolished both the Na(+) current (I(Na)) and a 75-kD gamma-ENaC fragment at the cell surface stimulated by CAP2. Replacement of gamma-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I(Na) and the 75-kD gamma-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in gamma-ENaC.

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Effect of α-R205A/R231A, β-, and γ-ENaC mutant on INa and fragments stimulated by CAP2. (A and B) Surface biotinylated α-ENaC N-terminal and C-terminal fragments were visualized by Western blot analysis using anti-HA or anti-V5 monoclonal antibodies. (A) Many nonspecific (*, NS) bands evident in film overexposed to reveal the fate of the furin (32 kD) and novel fragments (19 and 17 kD). FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-R205A/R231A, β-, and γ-ENaC alone; lane 5, α-R205A/R231A, β-, and γ-ENaC plus CAP2. (C) CAP2-mediated INa of WT or mutant channels were measured as described above. Batches of oocytes were extracted from five different frogs (n = 31). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test.
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fig7: Effect of α-R205A/R231A, β-, and γ-ENaC mutant on INa and fragments stimulated by CAP2. (A and B) Surface biotinylated α-ENaC N-terminal and C-terminal fragments were visualized by Western blot analysis using anti-HA or anti-V5 monoclonal antibodies. (A) Many nonspecific (*, NS) bands evident in film overexposed to reveal the fate of the furin (32 kD) and novel fragments (19 and 17 kD). FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-R205A/R231A, β-, and γ-ENaC alone; lane 5, α-R205A/R231A, β-, and γ-ENaC plus CAP2. (C) CAP2-mediated INa of WT or mutant channels were measured as described above. Batches of oocytes were extracted from five different frogs (n = 31). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test.

Mentions: Previous findings by Hughey et al. (2003) ascribed HA-NT α- and γ-ENaC fragments (α: 30 kD; γ: 18 kD) and complementary V5-CT α- and γ-ENaC fragments (α: 65 kD; γ: 75 kD) to cleavage by furin family proprotein convertases (Hughey et al., 2003). Prevention of these fragments by mutagenesis of the furin consensus cleavage sites diminished basal proteolytic activation of ENaC (Hughey et al., 2003, 2004). Because CAP2 consistently increased the intensity bands corresponding to α- and γ-ENaC furin fragments, we asked if this apparent enhanced cleavage contributed to CAP2 stimulation of basal INa. First, we compared the effects of CAP2 expression on WT and ENaC channels mutagenized to prevent cleavage by convertases. Although furin fragments were not prominent in every control (WT ENaC alone) experiment, coexpression with CAP2 reliably increased intensity of the HA-NT 32-kD and V5-CT 66-kD bands (furin fragments), and induced the novel HA-NT bands of 17, 19 (Fig. 7, A and B, compare lanes 2 and 3), and 82 kD (Fig. 3 A). For example, in the gel shown, the 32-kD HA-NT band (furin fragment) was hardly noticeable above background in the surface pool of control oocytes (WT ENaC alone) (Fig. 7 A, lane 2). This band is similarly indistinct in α-R205A/R231A, β-, and γ-ENaC–expressing oocytes (Fig. 7 A, lane 4). CAP2 coexpression with WT ENaC robustly increased the intensity of the 32-kD band, as well as generating the novel 17- and 19-kD bands (Fig. 7 A, lane 3). However, this effect of CAP2 coexpression was not seen in oocytes expressing α-R205A/R231A, β-, and γ-ENaC (Fig. 7 A, lane 5). Although Western blot sensitivity does not permit the presence of fragments to be ruled out, it is clear even from overexposed gels that CAP2 has minimal effects on the 32-kD band (taking into account background staining in the overexposed gel and slightly unequal loading of the lanes). The 66-kD V5-CT fragment was visible at the surface pool of WT ENaC eggs and enhanced when CAP2 was coexpressed; however, this fragment was not detectable in oocytes expressing α-R205A/R231A, β-, and γ-ENaC channels with/without CAP2 (Fig. 7 B). Not only did coexpression of CAP2 with α-R205A/R231A, β-, and γ-ENaC channels not increase the furin fragments significantly, but it also did not generate the smaller novel ∼17- and ∼19-kD α-ENaC N-terminal fragments seen in WT ENaC plus CAP2 experiments. (Fig. 7 A, lane 5 vs. lane 3). Nonetheless, the fold increase ratio of INa due CAP2 coexpression with the mutant channels was very similar (∼2.2-fold) to that of WT channels (∼2.0-fold) (Fig. 7 C) despite the absence of CAP2-induced fragments of α-ENaC (Fig. 7, A and B). Importantly, the elevated basal INa of oocytes coexpressing α-R205A/R231A, β, and γ-ENaC with CAP2 was unresponsive to exogenous trypsin, suggesting that CAP2 achieved full proteolytic activation of these mutant channels (Fig. 7 C). Basal INa of oocytes expressing α-R205A/R231A, β, and γ-ENaC was significantly smaller than INa of oocytes expressing WT channels, but responded briskly to trypsin, as reported by Hughey et al. (2004). These results do not support a strong correlation between apparent increased generation of α-ENaC furin fragments by CAP2 and stimulated INa. The elimination of the other CAP2-induced fragments by mutating the furin sites in α-ENaC suggests that they may have been generated secondarily to cleavage at the furin sites.


ENaC proteolytic regulation by channel-activating protease 2.

García-Caballero A, Dang Y, He H, Stutts MJ - J. Gen. Physiol. (2008)

Effect of α-R205A/R231A, β-, and γ-ENaC mutant on INa and fragments stimulated by CAP2. (A and B) Surface biotinylated α-ENaC N-terminal and C-terminal fragments were visualized by Western blot analysis using anti-HA or anti-V5 monoclonal antibodies. (A) Many nonspecific (*, NS) bands evident in film overexposed to reveal the fate of the furin (32 kD) and novel fragments (19 and 17 kD). FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-R205A/R231A, β-, and γ-ENaC alone; lane 5, α-R205A/R231A, β-, and γ-ENaC plus CAP2. (C) CAP2-mediated INa of WT or mutant channels were measured as described above. Batches of oocytes were extracted from five different frogs (n = 31). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test.
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Related In: Results  -  Collection

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fig7: Effect of α-R205A/R231A, β-, and γ-ENaC mutant on INa and fragments stimulated by CAP2. (A and B) Surface biotinylated α-ENaC N-terminal and C-terminal fragments were visualized by Western blot analysis using anti-HA or anti-V5 monoclonal antibodies. (A) Many nonspecific (*, NS) bands evident in film overexposed to reveal the fate of the furin (32 kD) and novel fragments (19 and 17 kD). FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-R205A/R231A, β-, and γ-ENaC alone; lane 5, α-R205A/R231A, β-, and γ-ENaC plus CAP2. (C) CAP2-mediated INa of WT or mutant channels were measured as described above. Batches of oocytes were extracted from five different frogs (n = 31). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test.
Mentions: Previous findings by Hughey et al. (2003) ascribed HA-NT α- and γ-ENaC fragments (α: 30 kD; γ: 18 kD) and complementary V5-CT α- and γ-ENaC fragments (α: 65 kD; γ: 75 kD) to cleavage by furin family proprotein convertases (Hughey et al., 2003). Prevention of these fragments by mutagenesis of the furin consensus cleavage sites diminished basal proteolytic activation of ENaC (Hughey et al., 2003, 2004). Because CAP2 consistently increased the intensity bands corresponding to α- and γ-ENaC furin fragments, we asked if this apparent enhanced cleavage contributed to CAP2 stimulation of basal INa. First, we compared the effects of CAP2 expression on WT and ENaC channels mutagenized to prevent cleavage by convertases. Although furin fragments were not prominent in every control (WT ENaC alone) experiment, coexpression with CAP2 reliably increased intensity of the HA-NT 32-kD and V5-CT 66-kD bands (furin fragments), and induced the novel HA-NT bands of 17, 19 (Fig. 7, A and B, compare lanes 2 and 3), and 82 kD (Fig. 3 A). For example, in the gel shown, the 32-kD HA-NT band (furin fragment) was hardly noticeable above background in the surface pool of control oocytes (WT ENaC alone) (Fig. 7 A, lane 2). This band is similarly indistinct in α-R205A/R231A, β-, and γ-ENaC–expressing oocytes (Fig. 7 A, lane 4). CAP2 coexpression with WT ENaC robustly increased the intensity of the 32-kD band, as well as generating the novel 17- and 19-kD bands (Fig. 7 A, lane 3). However, this effect of CAP2 coexpression was not seen in oocytes expressing α-R205A/R231A, β-, and γ-ENaC (Fig. 7 A, lane 5). Although Western blot sensitivity does not permit the presence of fragments to be ruled out, it is clear even from overexposed gels that CAP2 has minimal effects on the 32-kD band (taking into account background staining in the overexposed gel and slightly unequal loading of the lanes). The 66-kD V5-CT fragment was visible at the surface pool of WT ENaC eggs and enhanced when CAP2 was coexpressed; however, this fragment was not detectable in oocytes expressing α-R205A/R231A, β-, and γ-ENaC channels with/without CAP2 (Fig. 7 B). Not only did coexpression of CAP2 with α-R205A/R231A, β-, and γ-ENaC channels not increase the furin fragments significantly, but it also did not generate the smaller novel ∼17- and ∼19-kD α-ENaC N-terminal fragments seen in WT ENaC plus CAP2 experiments. (Fig. 7 A, lane 5 vs. lane 3). Nonetheless, the fold increase ratio of INa due CAP2 coexpression with the mutant channels was very similar (∼2.2-fold) to that of WT channels (∼2.0-fold) (Fig. 7 C) despite the absence of CAP2-induced fragments of α-ENaC (Fig. 7, A and B). Importantly, the elevated basal INa of oocytes coexpressing α-R205A/R231A, β, and γ-ENaC with CAP2 was unresponsive to exogenous trypsin, suggesting that CAP2 achieved full proteolytic activation of these mutant channels (Fig. 7 C). Basal INa of oocytes expressing α-R205A/R231A, β, and γ-ENaC was significantly smaller than INa of oocytes expressing WT channels, but responded briskly to trypsin, as reported by Hughey et al. (2004). These results do not support a strong correlation between apparent increased generation of α-ENaC furin fragments by CAP2 and stimulated INa. The elimination of the other CAP2-induced fragments by mutating the furin sites in α-ENaC suggests that they may have been generated secondarily to cleavage at the furin sites.

Bottom Line: Potential therapies for disorders of Na(+) absorption require better understanding of ENaC regulation.Replacement of gamma-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I(Na) and the 75-kD gamma-ENaC fragment.These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in gamma-ENaC.

View Article: PubMed Central - PubMed

Affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC 27599, USA. acaballe@med.unc.edu

ABSTRACT
Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na(+) absorption across epithelial surfaces throughout the body. Excessive Na(+) absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na(+) absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (alpha-K561, beta-R503, and gamma-R515). Sites in alpha-ENaC at K149/R164/K169/R177 and furin-consensus sites in alpha-ENaC (R205/R231) and gamma-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in gamma-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (alpha,beta,gammaR138A/Q) completely abolished both the Na(+) current (I(Na)) and a 75-kD gamma-ENaC fragment at the cell surface stimulated by CAP2. Replacement of gamma-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I(Na) and the 75-kD gamma-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in gamma-ENaC.

Show MeSH
Related in: MedlinePlus