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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-178-181 QQQQ or α-, β-, and γ-178-181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutants on INa stimulated by CAP2 and CAP1. (A) CAP2 induced INa of WT and 178–181 QQQQ ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (B) Western blot of surface pool of WT and 178–181 QQQQ γ-ENaC mutant channels. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-178-181 QQQQ ENaC alone; lane 5, α-, β-, and γ-178-181 QQQQ ENaC plus CAP2. A representative experiment is shown (n = 3). (C) CAP2 and CAP1 induced INa of WT and 178–181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, difference P < 0.05. Statistical significance was determined using ANOVA.
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fig9: Effect of α-, β-, and γ-178-181 QQQQ or α-, β-, and γ-178-181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutants on INa stimulated by CAP2 and CAP1. (A) CAP2 induced INa of WT and 178–181 QQQQ ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (B) Western blot of surface pool of WT and 178–181 QQQQ γ-ENaC mutant channels. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-178-181 QQQQ ENaC alone; lane 5, α-, β-, and γ-178-181 QQQQ ENaC plus CAP2. A representative experiment is shown (n = 3). (C) CAP2 and CAP1 induced INa of WT and 178–181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, difference P < 0.05. Statistical significance was determined using ANOVA.

Mentions: To determine if the polybasic tract 178–181 RKRK in rat γ-ENaC was important for CAP2 stimulation of rat ENaC, we replaced 178–181 RKRK with 178–181 QQQQ and determined the effect of CAP2 coexpression on INa and rat γ-ENaC fragments. We found that CAP2-stimulated INa in oocytes expressing this quadruple γ-ENaC mutant were indistinguishable from INa activated by CAP2 coexpressed with WT ENaC (Fig. 9 A). In addition, the fragment pattern induced by CAP2 in either WT or mutant γ-ENaC was identical (Fig. 9 B). These data demonstrate that CAP2 does not require the cluster of arginine and lysines (178–181) to activate rat ENaC, in contrast to the effect of CAP1 on mouse ENaC (Bruns et al., 2007). Interestingly, in our hands, CAP1/prostasin coexpression stimulated the QQQQ mutant as well as WT ENaC (threefold for WT and 2.8-fold for γ-ENaC 178–181 QQQQ; not depicted). Like Bruns et al. (2007), we conjectured that CAP2 was able to cleave the QQQQ mutant at nearby basic residues, and we therefore studied the effect of additional mutations (178–181 QQQQ plus K185Q, K189Q, K200Q, and K201Q). Both CAP1 and CAP2 stimulated basal currents of these mutant channels by 2.2- and 1.8-fold, respectively, and the stimulated basal INa was not further increased by trypsin (Fig. 9 C). Thus, other than the γ-ENaC furin site, we did not locate a second site containing basic residues that was required for stimulation of ENaC by CAP1 or CAP2 coexpression.


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-178-181 QQQQ or α-, β-, and γ-178-181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutants on INa stimulated by CAP2 and CAP1. (A) CAP2 induced INa of WT and 178–181 QQQQ ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (B) Western blot of surface pool of WT and 178–181 QQQQ γ-ENaC mutant channels. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-178-181 QQQQ ENaC alone; lane 5, α-, β-, and γ-178-181 QQQQ ENaC plus CAP2. A representative experiment is shown (n = 3). (C) CAP2 and CAP1 induced INa of WT and 178–181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, difference P < 0.05. Statistical significance was determined using ANOVA.
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Related In: Results  -  Collection

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fig9: Effect of α-, β-, and γ-178-181 QQQQ or α-, β-, and γ-178-181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutants on INa stimulated by CAP2 and CAP1. (A) CAP2 induced INa of WT and 178–181 QQQQ ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, P < 0.0001. Statistical significance was determined using an unpaired Student's t test. (B) Western blot of surface pool of WT and 178–181 QQQQ γ-ENaC mutant channels. Lane 1, uninjected eggs; lane 2, WT ENaC alone; lane 3, WT ENaC plus CAP2; lane 4, α-, β-, and γ-178-181 QQQQ ENaC alone; lane 5, α-, β-, and γ-178-181 QQQQ ENaC plus CAP2. A representative experiment is shown (n = 3). (C) CAP2 and CAP1 induced INa of WT and 178–181 QQQQ, K185Q, K189Q, K200Q, and K201Q ENaC mutant channels. Amiloride-sensitive currents were measured as described in Fig. 1. Batches of oocytes were extracted from three different frogs (n = 12). Results are expressed as the means ± SE. * and **, difference P < 0.05. Statistical significance was determined using ANOVA.
Mentions: To determine if the polybasic tract 178–181 RKRK in rat γ-ENaC was important for CAP2 stimulation of rat ENaC, we replaced 178–181 RKRK with 178–181 QQQQ and determined the effect of CAP2 coexpression on INa and rat γ-ENaC fragments. We found that CAP2-stimulated INa in oocytes expressing this quadruple γ-ENaC mutant were indistinguishable from INa activated by CAP2 coexpressed with WT ENaC (Fig. 9 A). In addition, the fragment pattern induced by CAP2 in either WT or mutant γ-ENaC was identical (Fig. 9 B). These data demonstrate that CAP2 does not require the cluster of arginine and lysines (178–181) to activate rat ENaC, in contrast to the effect of CAP1 on mouse ENaC (Bruns et al., 2007). Interestingly, in our hands, CAP1/prostasin coexpression stimulated the QQQQ mutant as well as WT ENaC (threefold for WT and 2.8-fold for γ-ENaC 178–181 QQQQ; not depicted). Like Bruns et al. (2007), we conjectured that CAP2 was able to cleave the QQQQ mutant at nearby basic residues, and we therefore studied the effect of additional mutations (178–181 QQQQ plus K185Q, K189Q, K200Q, and K201Q). Both CAP1 and CAP2 stimulated basal currents of these mutant channels by 2.2- and 1.8-fold, respectively, and the stimulated basal INa was not further increased by trypsin (Fig. 9 C). Thus, other than the γ-ENaC furin site, we did not locate a second site containing basic residues that was required for stimulation of ENaC by CAP1 or CAP2 coexpression.

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