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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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Cleavage of α-, β-, and γ-ENaC–tagged subunits by WT but not inactive CAP2 S387A in oocytes. WT α-, β-, and γ-ENaC–double tagged (HA-NT/V5-CT) and α-, β-, or γ-ENaC–untagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes, and Western blots analysis were conducted using anti-HA (A) and anti-V5 (B) monoclonal antibodies. Western blots of surface biotinylated and total pools of α-ENaC HA-NT (C) and γ-ENaC V5-CT (D) fragments caused by WT but not inactive CAP2 (CAP2 S387A). Actin expression was detected with an anti-actin monoclonal antibody as control (D). A β-ENaC 90-kD HA-NT fragment was detected at the surface pool (not depicted). NS, nonspecific. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (E) Linear diagram of ENaC residues cleaved by CAP2. NH2, amino terminus; COOH, carboxy terminus; TM1, transmembrane domain 1; TM2, transmembrane domain 2.
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fig3: Cleavage of α-, β-, and γ-ENaC–tagged subunits by WT but not inactive CAP2 S387A in oocytes. WT α-, β-, and γ-ENaC–double tagged (HA-NT/V5-CT) and α-, β-, or γ-ENaC–untagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes, and Western blots analysis were conducted using anti-HA (A) and anti-V5 (B) monoclonal antibodies. Western blots of surface biotinylated and total pools of α-ENaC HA-NT (C) and γ-ENaC V5-CT (D) fragments caused by WT but not inactive CAP2 (CAP2 S387A). Actin expression was detected with an anti-actin monoclonal antibody as control (D). A β-ENaC 90-kD HA-NT fragment was detected at the surface pool (not depicted). NS, nonspecific. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (E) Linear diagram of ENaC residues cleaved by CAP2. NH2, amino terminus; COOH, carboxy terminus; TM1, transmembrane domain 1; TM2, transmembrane domain 2.

Mentions: The observation that trypsin and MTSET do not stimulate the INa of ENaC coexpressed with active CAP2 indicates that ENaC at the surface of these oocytes are maximally proteolytically stimulated to a high PO. To identify ENaC cleavage(s) in the presence of CAP2 that could increase PO, we examined the fragmentation pattern of ENaC when coexpressed with CAP2 using HA-NT– and V5-CT–tagged subunits (Hughey et al., 2003) (Fig. 3). With ENaC alone, we typically observed a pattern of fragments similar to that reported (Hughey et al., 2003). Western blots stained for epitope-tagged α-ENaC or γ-ENaC each variably demonstrated fragments (α-ENaC HA-NT, ∼32 kD; γ-ENaC HA-NT, ∼18 kD; α-ENaC V5-CT, ∼66 kD; γ-ENaC V5-CT, ∼75 kD), consistent with cleavage at identified consensus cleavage sites for members of the protein convertase family, whereas Western blots of β-ENaC, as reported previously, did not indicate cleavage under basal conditions (Hughey et al., 2003).


ENaC proteolytic regulation by channel-activating protease 2.

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

Cleavage of α-, β-, and γ-ENaC–tagged subunits by WT but not inactive CAP2 S387A in oocytes. WT α-, β-, and γ-ENaC–double tagged (HA-NT/V5-CT) and α-, β-, or γ-ENaC–untagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes, and Western blots analysis were conducted using anti-HA (A) and anti-V5 (B) monoclonal antibodies. Western blots of surface biotinylated and total pools of α-ENaC HA-NT (C) and γ-ENaC V5-CT (D) fragments caused by WT but not inactive CAP2 (CAP2 S387A). Actin expression was detected with an anti-actin monoclonal antibody as control (D). A β-ENaC 90-kD HA-NT fragment was detected at the surface pool (not depicted). NS, nonspecific. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (E) Linear diagram of ENaC residues cleaved by CAP2. NH2, amino terminus; COOH, carboxy terminus; TM1, transmembrane domain 1; TM2, transmembrane domain 2.
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fig3: Cleavage of α-, β-, and γ-ENaC–tagged subunits by WT but not inactive CAP2 S387A in oocytes. WT α-, β-, and γ-ENaC–double tagged (HA-NT/V5-CT) and α-, β-, or γ-ENaC–untagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes, and Western blots analysis were conducted using anti-HA (A) and anti-V5 (B) monoclonal antibodies. Western blots of surface biotinylated and total pools of α-ENaC HA-NT (C) and γ-ENaC V5-CT (D) fragments caused by WT but not inactive CAP2 (CAP2 S387A). Actin expression was detected with an anti-actin monoclonal antibody as control (D). A β-ENaC 90-kD HA-NT fragment was detected at the surface pool (not depicted). NS, nonspecific. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (E) Linear diagram of ENaC residues cleaved by CAP2. NH2, amino terminus; COOH, carboxy terminus; TM1, transmembrane domain 1; TM2, transmembrane domain 2.
Mentions: The observation that trypsin and MTSET do not stimulate the INa of ENaC coexpressed with active CAP2 indicates that ENaC at the surface of these oocytes are maximally proteolytically stimulated to a high PO. To identify ENaC cleavage(s) in the presence of CAP2 that could increase PO, we examined the fragmentation pattern of ENaC when coexpressed with CAP2 using HA-NT– and V5-CT–tagged subunits (Hughey et al., 2003) (Fig. 3). With ENaC alone, we typically observed a pattern of fragments similar to that reported (Hughey et al., 2003). Western blots stained for epitope-tagged α-ENaC or γ-ENaC each variably demonstrated fragments (α-ENaC HA-NT, ∼32 kD; γ-ENaC HA-NT, ∼18 kD; α-ENaC V5-CT, ∼66 kD; γ-ENaC V5-CT, ∼75 kD), consistent with cleavage at identified consensus cleavage sites for members of the protein convertase family, whereas Western blots of β-ENaC, as reported previously, did not indicate cleavage under basal conditions (Hughey et al., 2003).

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