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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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Catalytic activity of CAP2 is required to stimulate ENaC. (A) INa stimulated by CAP2 is sensitive to aprotinin. Eggs coexpressing ENaC plus CAP2 were preincubated or not with 50 μg/ml aprotinin overnight (n = 18). ENaC WT plus CAP2 versus ENaC WT plus CAP2 plus aprotinin. *, P < 0.0001. (B) Inactive CAP2 s387a (CAP2s387a) does not activate ENaC. Amiloride-sensitive INa stimulated by WT but not by inactive CAP2 (n = 30). (A and B) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. *, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Inactive CAP2 is expressed as a full-length precursor. Western blots of WT and inactive express-tagged CAP2. Representative experiment is shown.
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fig2: Catalytic activity of CAP2 is required to stimulate ENaC. (A) INa stimulated by CAP2 is sensitive to aprotinin. Eggs coexpressing ENaC plus CAP2 were preincubated or not with 50 μg/ml aprotinin overnight (n = 18). ENaC WT plus CAP2 versus ENaC WT plus CAP2 plus aprotinin. *, P < 0.0001. (B) Inactive CAP2 s387a (CAP2s387a) does not activate ENaC. Amiloride-sensitive INa stimulated by WT but not by inactive CAP2 (n = 30). (A and B) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. *, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Inactive CAP2 is expressed as a full-length precursor. Western blots of WT and inactive express-tagged CAP2. Representative experiment is shown.

Mentions: To establish cleavage of ENaC in the presence of CAP2 as a potential mechanism for stimulation of INa, we next asked if CAP2 catalytic activity was required for stimulation of coexpressed ENaC. We addressed this question in two ways. Because CAP2-mediated proteolysis is inhibited by aprotinin, we asked if aprotinin blocked CAP2 regulation of ENaC. Overnight incubation of oocytes with 50 μg/ml aprotinin prevented the stimulation of currents typically seen from coexpressing ENaC and CAP2 (Fig. 2 A). This result does not rule out indirect action of CAP2 on ENaC through another aprotinin-sensitive protease, as has been proposed for CAP1 (Andreasen et al., 2006). Therefore, we constructed an inactive CAP2 control by mutating serine 387 in its catalytic triad (CAP2s387a) and tested its effect on ENaC. This mutation alters the HDS triad at the catalytic site of CAP2 resulting in inactivation (Andreasen et al., 2006). As shown in Fig. 2 B, mutation of the catalytic triad of CAP2 abolished the protease's ability to stimulate basal INa.


ENaC proteolytic regulation by channel-activating protease 2.

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

Catalytic activity of CAP2 is required to stimulate ENaC. (A) INa stimulated by CAP2 is sensitive to aprotinin. Eggs coexpressing ENaC plus CAP2 were preincubated or not with 50 μg/ml aprotinin overnight (n = 18). ENaC WT plus CAP2 versus ENaC WT plus CAP2 plus aprotinin. *, P < 0.0001. (B) Inactive CAP2 s387a (CAP2s387a) does not activate ENaC. Amiloride-sensitive INa stimulated by WT but not by inactive CAP2 (n = 30). (A and B) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. *, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Inactive CAP2 is expressed as a full-length precursor. Western blots of WT and inactive express-tagged CAP2. Representative experiment is shown.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571966&req=5

fig2: Catalytic activity of CAP2 is required to stimulate ENaC. (A) INa stimulated by CAP2 is sensitive to aprotinin. Eggs coexpressing ENaC plus CAP2 were preincubated or not with 50 μg/ml aprotinin overnight (n = 18). ENaC WT plus CAP2 versus ENaC WT plus CAP2 plus aprotinin. *, P < 0.0001. (B) Inactive CAP2 s387a (CAP2s387a) does not activate ENaC. Amiloride-sensitive INa stimulated by WT but not by inactive CAP2 (n = 30). (A and B) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. *, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (C) Inactive CAP2 is expressed as a full-length precursor. Western blots of WT and inactive express-tagged CAP2. Representative experiment is shown.
Mentions: To establish cleavage of ENaC in the presence of CAP2 as a potential mechanism for stimulation of INa, we next asked if CAP2 catalytic activity was required for stimulation of coexpressed ENaC. We addressed this question in two ways. Because CAP2-mediated proteolysis is inhibited by aprotinin, we asked if aprotinin blocked CAP2 regulation of ENaC. Overnight incubation of oocytes with 50 μg/ml aprotinin prevented the stimulation of currents typically seen from coexpressing ENaC and CAP2 (Fig. 2 A). This result does not rule out indirect action of CAP2 on ENaC through another aprotinin-sensitive protease, as has been proposed for CAP1 (Andreasen et al., 2006). Therefore, we constructed an inactive CAP2 control by mutating serine 387 in its catalytic triad (CAP2s387a) and tested its effect on ENaC. This mutation alters the HDS triad at the catalytic site of CAP2 resulting in inactivation (Andreasen et al., 2006). As shown in Fig. 2 B, mutation of the catalytic triad of CAP2 abolished the protease's ability to stimulate basal INa.

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