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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 replacement of γ-ENaC R138 (P1) by alanine, lysine, or glutamine on INa and fragments stimulated by CAP2. (A) CAP2 effect on INa of WT and α-, β-, and γ-R138A or α-, β-, and γ-R138K or α-, β-, and γ-R138Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 18). Results are expressed as the means ± SE. * and **, P < 0.0001. (B) Western blots of total whole cell extracts. CAP2-induced fragment pattern of WT and γ-ENaC mutant channels (top) and actin as loading control (bottom). Lane 1, uninjected eggs; lane 2, WT ENaC plus CAP2; lane 3, α-, β-, and γ-R138A ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC plus CAP2; lane 5, α-, β-, and γ-R138Q ENaC plus CAP2. A representative experiment is shown (n = 3). (C) Western blots of surface biotinylated (top), total γ-ENaC pools (bottom), and actin. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC alone; lane 5, α-, β-, and γ-R138K ENaC plus CAP2. A representative experiment is shown (n = 3).
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fig10: Effect of replacement of γ-ENaC R138 (P1) by alanine, lysine, or glutamine on INa and fragments stimulated by CAP2. (A) CAP2 effect on INa of WT and α-, β-, and γ-R138A or α-, β-, and γ-R138K or α-, β-, and γ-R138Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 18). Results are expressed as the means ± SE. * and **, P < 0.0001. (B) Western blots of total whole cell extracts. CAP2-induced fragment pattern of WT and γ-ENaC mutant channels (top) and actin as loading control (bottom). Lane 1, uninjected eggs; lane 2, WT ENaC plus CAP2; lane 3, α-, β-, and γ-R138A ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC plus CAP2; lane 5, α-, β-, and γ-R138Q ENaC plus CAP2. A representative experiment is shown (n = 3). (C) Western blots of surface biotinylated (top), total γ-ENaC pools (bottom), and actin. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC alone; lane 5, α-, β-, and γ-R138K ENaC plus CAP2. A representative experiment is shown (n = 3).

Mentions: Our results with previously validated furin mutants indicate that cleavage at R138 in γ-ENaC is largely responsible for the stimulation of INa by coexpressed CAP2. Because this residue occupies the critical P1 site for furin and similar convertases, we next asked if CAP2 was actually cleaving after R138 or if CAP2 stimulated furin or another convertase to cleave there. The minimal P4-P1 recognition sequence for furin is R/K-X-X-R, and the requirement for arginine at P1 is stringent (Thomas, 2002; Wheatley and Holyoak, 2007). Interestingly, recent crystallization and other work reveal a similar, but not identical, optimal sequence for matriptase binding and cleavage, R-X-X-K/R (Friedrich et al., 2002; Netzel-Arnett et al., 2003; Desilets et al., 2006). Furthermore, a similar P4-P1 preferred sequence for CAP1/prostasin, R/K-H/R/K-X-R/K, was identified by screening peptide libraries (Shipway et al., 2004). These published observations indicate that CAP1 and CAP3 prefer an arginine, but tolerate a lysine in the P1 position. We speculated that if CAP2 shared this relaxed stringency at P1, CAP2 coexpression would stimulate the furin-resistant ENaC mutant α-, β-, and γ-R138K. We also tested the effect of CAP2 coexpression on α-, β-, and γ-R138Q ENaC, in which γ residue 138 should not be a substrate for either furin or CAP2. We found that basal INa of ENaC containing γ-R138A or γ-R138Q was not increased by coexpressed CAP2, but was robustly stimulated by exogenous trypsin (Fig. 10 A). In contrast, when ENaC contained γ-R138K, CAP2 coexpression significantly increased basal INa. Importantly, trypsin did not further stimulate INa of oocytes expressing α-, β-, and γ-R138K plus CAP2, indicating that ENaC in these oocytes had already been proteolytically activated. Thus, CAP2 stimulated ENaC when the critical 138 (P1) residue in the γ-subunit was a lysine. These results strongly suggest that cleavage induced by CAP2 at R138 in γ-ENaC is responsible for CAP2 stimulation of INa.


ENaC proteolytic regulation by channel-activating protease 2.

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

Effect of replacement of γ-ENaC R138 (P1) by alanine, lysine, or glutamine on INa and fragments stimulated by CAP2. (A) CAP2 effect on INa of WT and α-, β-, and γ-R138A or α-, β-, and γ-R138K or α-, β-, and γ-R138Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 18). Results are expressed as the means ± SE. * and **, P < 0.0001. (B) Western blots of total whole cell extracts. CAP2-induced fragment pattern of WT and γ-ENaC mutant channels (top) and actin as loading control (bottom). Lane 1, uninjected eggs; lane 2, WT ENaC plus CAP2; lane 3, α-, β-, and γ-R138A ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC plus CAP2; lane 5, α-, β-, and γ-R138Q ENaC plus CAP2. A representative experiment is shown (n = 3). (C) Western blots of surface biotinylated (top), total γ-ENaC pools (bottom), and actin. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC alone; lane 5, α-, β-, and γ-R138K ENaC plus CAP2. A representative experiment is shown (n = 3).
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fig10: Effect of replacement of γ-ENaC R138 (P1) by alanine, lysine, or glutamine on INa and fragments stimulated by CAP2. (A) CAP2 effect on INa of WT and α-, β-, and γ-R138A or α-, β-, and γ-R138K or α-, β-, and γ-R138Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 18). Results are expressed as the means ± SE. * and **, P < 0.0001. (B) Western blots of total whole cell extracts. CAP2-induced fragment pattern of WT and γ-ENaC mutant channels (top) and actin as loading control (bottom). Lane 1, uninjected eggs; lane 2, WT ENaC plus CAP2; lane 3, α-, β-, and γ-R138A ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC plus CAP2; lane 5, α-, β-, and γ-R138Q ENaC plus CAP2. A representative experiment is shown (n = 3). (C) Western blots of surface biotinylated (top), total γ-ENaC pools (bottom), and actin. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-R138K ENaC alone; lane 5, α-, β-, and γ-R138K ENaC plus CAP2. A representative experiment is shown (n = 3).
Mentions: Our results with previously validated furin mutants indicate that cleavage at R138 in γ-ENaC is largely responsible for the stimulation of INa by coexpressed CAP2. Because this residue occupies the critical P1 site for furin and similar convertases, we next asked if CAP2 was actually cleaving after R138 or if CAP2 stimulated furin or another convertase to cleave there. The minimal P4-P1 recognition sequence for furin is R/K-X-X-R, and the requirement for arginine at P1 is stringent (Thomas, 2002; Wheatley and Holyoak, 2007). Interestingly, recent crystallization and other work reveal a similar, but not identical, optimal sequence for matriptase binding and cleavage, R-X-X-K/R (Friedrich et al., 2002; Netzel-Arnett et al., 2003; Desilets et al., 2006). Furthermore, a similar P4-P1 preferred sequence for CAP1/prostasin, R/K-H/R/K-X-R/K, was identified by screening peptide libraries (Shipway et al., 2004). These published observations indicate that CAP1 and CAP3 prefer an arginine, but tolerate a lysine in the P1 position. We speculated that if CAP2 shared this relaxed stringency at P1, CAP2 coexpression would stimulate the furin-resistant ENaC mutant α-, β-, and γ-R138K. We also tested the effect of CAP2 coexpression on α-, β-, and γ-R138Q ENaC, in which γ residue 138 should not be a substrate for either furin or CAP2. We found that basal INa of ENaC containing γ-R138A or γ-R138Q was not increased by coexpressed CAP2, but was robustly stimulated by exogenous trypsin (Fig. 10 A). In contrast, when ENaC contained γ-R138K, CAP2 coexpression significantly increased basal INa. Importantly, trypsin did not further stimulate INa of oocytes expressing α-, β-, and γ-R138K plus CAP2, indicating that ENaC in these oocytes had already been proteolytically activated. Thus, CAP2 stimulated ENaC when the critical 138 (P1) residue in the γ-subunit was a lysine. These results strongly suggest that cleavage induced by CAP2 at R138 in γ-ENaC is responsible for CAP2 stimulation of 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