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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-, β-, and γ-R138A-ENaC mutant on INa and fragments stimulated by CAP2. (A) (Top) Surface biotinylated γ-ENaC CT 75-kD fragment induced by CAP2 was visualized by Western blot analysis using an anti-V5 monoclonal antibody. (Bottom) Total whole cell lysates (input 4%) were probed for actin as loading control. FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138A ENaC alone; lane 5, α-, β-, and γ-R138A ENaC plus CAP2. A representative experiment is shown (n = 3). (B) CAP2-mediated INa of WT and α-, β-, and γ-R138A mutant channels were measured as described in Results. Batches of oocytes were extracted from six different frogs (n = 36). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Semi-quantification of the 75-kD γ-ENaC surface fragment induced by CAP2 (IOD, integrated optic density; n = 4).
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fig8: Effect of α-, β-, and γ-R138A-ENaC mutant on INa and fragments stimulated by CAP2. (A) (Top) Surface biotinylated γ-ENaC CT 75-kD fragment induced by CAP2 was visualized by Western blot analysis using an anti-V5 monoclonal antibody. (Bottom) Total whole cell lysates (input 4%) were probed for actin as loading control. FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138A ENaC alone; lane 5, α-, β-, and γ-R138A ENaC plus CAP2. A representative experiment is shown (n = 3). (B) CAP2-mediated INa of WT and α-, β-, and γ-R138A mutant channels were measured as described in Results. Batches of oocytes were extracted from six different frogs (n = 36). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Semi-quantification of the 75-kD γ-ENaC surface fragment induced by CAP2 (IOD, integrated optic density; n = 4).

Mentions: We next tested the role of γ-ENaC's single furin consensus site (135–138 RKRR) in CAP2 stimulation by substituting alanine for the arginine at residue 138 in γ-ENaC (135–138 RKRA). This change in the critical P1 site in the furin consensus cleavage site is expected to eliminate cleavage by furin-like convertases, and indeed, γ-R138A in the surface pool appeared on Western blot predominantly as the full-length channel, with the ∼75-kD C-terminal band that has been termed a furin fragment nearly eliminated (Fig. 8 A, compare lanes 2 [WT γ] and 4 [γ-R138A]). For WT γ-ENaC at the surface of CAP2-expressing oocytes, a broad, ∼75-kD band was markedly increased in intensity (Figs. 8 A and 3 D). In ENaC containing γ-R138A, most staining represented the full-length subunit, and CAP2 coexpression produced no marked increase in the ∼75-kD band (Fig. 8 A, lanes 4 and 5). An ∼73-kD band was also visible in lysates of γ-furin mutants and enhanced by CAP2 coexpression. Densitometric quantification of the 75-kD fragment of WT γ-ENaC showed an ∼2.4-fold increase induced by CAP2 coexpression (Fig. 8 C), which correlates with a routinely two- to threefold increase in INa observed with CAP2 coexpression (Fig. 8 B). Basal INa and fold trypsin stimulation for ENaC containing γ-R138A was not much different from WT ENaC. However, CAP2 coexpression did not increase basal current of oocytes expressing α-, β-, and γ-R138A. Notably, subsequent exposure of the α-, β-, and γ-R138A plus CAP2 oocytes to trypsin stimulated INa by more than twofold. These results identify R138 in γ-ENaC as a residue required for γ-ENaC cleavage and stimulation of INa in the presence of CAP2.


ENaC proteolytic regulation by channel-activating protease 2.

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

Effect of α-, β-, and γ-R138A-ENaC mutant on INa and fragments stimulated by CAP2. (A) (Top) Surface biotinylated γ-ENaC CT 75-kD fragment induced by CAP2 was visualized by Western blot analysis using an anti-V5 monoclonal antibody. (Bottom) Total whole cell lysates (input 4%) were probed for actin as loading control. FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138A ENaC alone; lane 5, α-, β-, and γ-R138A ENaC plus CAP2. A representative experiment is shown (n = 3). (B) CAP2-mediated INa of WT and α-, β-, and γ-R138A mutant channels were measured as described in Results. Batches of oocytes were extracted from six different frogs (n = 36). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Semi-quantification of the 75-kD γ-ENaC surface fragment induced by CAP2 (IOD, integrated optic density; n = 4).
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fig8: Effect of α-, β-, and γ-R138A-ENaC mutant on INa and fragments stimulated by CAP2. (A) (Top) Surface biotinylated γ-ENaC CT 75-kD fragment induced by CAP2 was visualized by Western blot analysis using an anti-V5 monoclonal antibody. (Bottom) Total whole cell lysates (input 4%) were probed for actin as loading control. FL, full-length. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138A ENaC alone; lane 5, α-, β-, and γ-R138A ENaC plus CAP2. A representative experiment is shown (n = 3). (B) CAP2-mediated INa of WT and α-, β-, and γ-R138A mutant channels were measured as described in Results. Batches of oocytes were extracted from six different frogs (n = 36). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Semi-quantification of the 75-kD γ-ENaC surface fragment induced by CAP2 (IOD, integrated optic density; n = 4).
Mentions: We next tested the role of γ-ENaC's single furin consensus site (135–138 RKRR) in CAP2 stimulation by substituting alanine for the arginine at residue 138 in γ-ENaC (135–138 RKRA). This change in the critical P1 site in the furin consensus cleavage site is expected to eliminate cleavage by furin-like convertases, and indeed, γ-R138A in the surface pool appeared on Western blot predominantly as the full-length channel, with the ∼75-kD C-terminal band that has been termed a furin fragment nearly eliminated (Fig. 8 A, compare lanes 2 [WT γ] and 4 [γ-R138A]). For WT γ-ENaC at the surface of CAP2-expressing oocytes, a broad, ∼75-kD band was markedly increased in intensity (Figs. 8 A and 3 D). In ENaC containing γ-R138A, most staining represented the full-length subunit, and CAP2 coexpression produced no marked increase in the ∼75-kD band (Fig. 8 A, lanes 4 and 5). An ∼73-kD band was also visible in lysates of γ-furin mutants and enhanced by CAP2 coexpression. Densitometric quantification of the 75-kD fragment of WT γ-ENaC showed an ∼2.4-fold increase induced by CAP2 coexpression (Fig. 8 C), which correlates with a routinely two- to threefold increase in INa observed with CAP2 coexpression (Fig. 8 B). Basal INa and fold trypsin stimulation for ENaC containing γ-R138A was not much different from WT ENaC. However, CAP2 coexpression did not increase basal current of oocytes expressing α-, β-, and γ-R138A. Notably, subsequent exposure of the α-, β-, and γ-R138A plus CAP2 oocytes to trypsin stimulated INa by more than twofold. These results identify R138 in γ-ENaC as a residue required for γ-ENaC cleavage and stimulation of INa in the presence of CAP2.

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