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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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CAP2 cleaves α-, β-, and γ-ENaC at a conserved basic residue in the pre-M2 region. WT α-, β-, and γ-ENaC–untagged or –double tagged (HA-NT/V5-CT) or α-K561A, β-R503A, and γ-R515A ENaC–tagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes and Western blot analysis was conducted using anti-V5 monoclonal antibodies (A). Black arrows indicate full-length (FL) and cleaved ENaC subunits. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (B) α-, β-, and γ-ENaC human, mouse, and rat pre-M2 region sequences alignment. TMII, transmembrane 2.
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fig5: CAP2 cleaves α-, β-, and γ-ENaC at a conserved basic residue in the pre-M2 region. WT α-, β-, and γ-ENaC–untagged or –double tagged (HA-NT/V5-CT) or α-K561A, β-R503A, and γ-R515A ENaC–tagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes and Western blot analysis was conducted using anti-V5 monoclonal antibodies (A). Black arrows indicate full-length (FL) and cleaved ENaC subunits. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (B) α-, β-, and γ-ENaC human, mouse, and rat pre-M2 region sequences alignment. TMII, transmembrane 2.

Mentions: We first sought to identify specific residues necessary for CAP2-stimulated fragments in the pre-M2 regions of α-, β-, and γ-ENaC. We analyzed α-, β-, and γ-ENaC sequences corresponding to the extracellular loop at the pre-M2 region with a cleavage prediction model for CAP3/matriptase available at http://pops.csse.monash.edu.au/pops.html. Although there is no established model to predict cleavage sites for CAP2, we have observed that CAP2 and CAP3/matriptase generate an identical C terminal–fragmentation pattern in the α-ENaC subunit (not depicted). From this observation, we reasoned that these CAPs could share some substrate affinity. Based on the apparent molecular mass of the ∼15–17-kD C terminal fragments generated by CAP2 in all three subunits, we surveyed a stretch of 40 amino acids in the pre-M2 region of α-, β-, and γ-ENaC. Whereas multiple basic residues in this region had high predictive scores for matriptase cleavage, we noted an aligned basic residue at position K561 in α-ENaC, R503 in β-ENaC, and R515 in γ-ENaC that was conserved across all three subunits of human, mouse, and rat ENaCs (Fig. 5 B). When we replaced basic residues at this residue with alanines by site-directed mutagenesis, the appearance of the pre-M2 fragments with CAP2 coexpression was eliminated (Fig. 5 A). Interestingly, in the mutants there was a trend toward lighter intensity of bands associated with cleavage at the furin sites in α- and γ-ENaC. Mutagenesis of other basic residues (α-ENaC: K486A, K501A, R503A, K504A, K512A, R519A, K524A, K544A, R545A, K550A, and K556A; β-ENaC: K411A, R416A, R435A, K443A, K452A, R477A, R487A, K488A, and K492A; γ-ENaC: K504A and K510A) in the 80-residue stretch had no effects on CAP2-mediated fragments (not depicted).


ENaC proteolytic regulation by channel-activating protease 2.

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

CAP2 cleaves α-, β-, and γ-ENaC at a conserved basic residue in the pre-M2 region. WT α-, β-, and γ-ENaC–untagged or –double tagged (HA-NT/V5-CT) or α-K561A, β-R503A, and γ-R515A ENaC–tagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes and Western blot analysis was conducted using anti-V5 monoclonal antibodies (A). Black arrows indicate full-length (FL) and cleaved ENaC subunits. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (B) α-, β-, and γ-ENaC human, mouse, and rat pre-M2 region sequences alignment. TMII, transmembrane 2.
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fig5: CAP2 cleaves α-, β-, and γ-ENaC at a conserved basic residue in the pre-M2 region. WT α-, β-, and γ-ENaC–untagged or –double tagged (HA-NT/V5-CT) or α-K561A, β-R503A, and γ-R515A ENaC–tagged subunits (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. Total protein lysates were prepared from oocytes and Western blot analysis was conducted using anti-V5 monoclonal antibodies (A). Black arrows indicate full-length (FL) and cleaved ENaC subunits. Batches of oocytes were extracted from three different frogs. Representative experiments are shown (n = 3). (B) α-, β-, and γ-ENaC human, mouse, and rat pre-M2 region sequences alignment. TMII, transmembrane 2.
Mentions: We first sought to identify specific residues necessary for CAP2-stimulated fragments in the pre-M2 regions of α-, β-, and γ-ENaC. We analyzed α-, β-, and γ-ENaC sequences corresponding to the extracellular loop at the pre-M2 region with a cleavage prediction model for CAP3/matriptase available at http://pops.csse.monash.edu.au/pops.html. Although there is no established model to predict cleavage sites for CAP2, we have observed that CAP2 and CAP3/matriptase generate an identical C terminal–fragmentation pattern in the α-ENaC subunit (not depicted). From this observation, we reasoned that these CAPs could share some substrate affinity. Based on the apparent molecular mass of the ∼15–17-kD C terminal fragments generated by CAP2 in all three subunits, we surveyed a stretch of 40 amino acids in the pre-M2 region of α-, β-, and γ-ENaC. Whereas multiple basic residues in this region had high predictive scores for matriptase cleavage, we noted an aligned basic residue at position K561 in α-ENaC, R503 in β-ENaC, and R515 in γ-ENaC that was conserved across all three subunits of human, mouse, and rat ENaCs (Fig. 5 B). When we replaced basic residues at this residue with alanines by site-directed mutagenesis, the appearance of the pre-M2 fragments with CAP2 coexpression was eliminated (Fig. 5 A). Interestingly, in the mutants there was a trend toward lighter intensity of bands associated with cleavage at the furin sites in α- and γ-ENaC. Mutagenesis of other basic residues (α-ENaC: K486A, K501A, R503A, K504A, K512A, R519A, K524A, K544A, R545A, K550A, and K556A; β-ENaC: K411A, R416A, R435A, K443A, K452A, R477A, R487A, K488A, and K492A; γ-ENaC: K504A and K510A) in the 80-residue stretch had no effects on CAP2-mediated fragments (not depicted).

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