Limits...
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.

Show MeSH

Related in: MedlinePlus

CAP2 stimulated α-R205A/R231A, β-, and γ-138K or α-, β-, and γ-138K plus 178–181 QQQQ but not α-R205A/R231A, β-, and γ-138A or α-, β-, and γ-138A plus 178–181 QQQQ mutant channels. (A) CAP2 induced INa of WT and α-R205A/R231A, β-, γ-, or α-R205A/R231A, β-, and γ-R138A or α-R205A/R231A, β-, and γ-R138K ENaC mutant channels (n = 12). (B) CAP2 induced INa of α-, β-, and γ-R138A, R178Q, K179Q, R180Q, and K181Q or α-, β-, and γ-R138K, R178Q, K179Q, R180Q, and K181Q 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig11: CAP2 stimulated α-R205A/R231A, β-, and γ-138K or α-, β-, and γ-138K plus 178–181 QQQQ but not α-R205A/R231A, β-, and γ-138A or α-, β-, and γ-138A plus 178–181 QQQQ mutant channels. (A) CAP2 induced INa of WT and α-R205A/R231A, β-, γ-, or α-R205A/R231A, β-, and γ-R138A or α-R205A/R231A, β-, and γ-R138K ENaC mutant channels (n = 12). (B) CAP2 induced INa of α-, β-, and γ-R138A, R178Q, K179Q, R180Q, and K181Q or α-, β-, and γ-R138K, R178Q, K179Q, R180Q, and K181Q 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.

Mentions: To further test the importance of γ-ENaC residue 138 in CAP2 stimulation of ENaC, we coexpressed either WT γ or γ-R138A or γ-R138K with α-R205A/R231A and WT β-ENaC. INa was stimulated by CAP2 coexpression only when an arginine (WT) or a lysine was present at position 138 in γ-ENaC (Fig. 11 A). We further isolated the action of CAP2 by coexpressing CAP2 with WT α- and β-ENaC combined with γ-178-181 QQQQ mutants that contained either alanine or lysine at γ-ENaC residue 138. These mutant γ-subunits are not expected to be cleaved by convertases acting at the γ-ENaC furin site, and they lack the polybasic tract implicated in CAP1 stimulation of mouse ENaC. CAP2 did not stimulate basal INa of ENaC with the double mutant γ-subunit containing alanine at residue 138; however, INa was markedly stimulated by subsequent trypsin (Fig. 11 B). In contrast, CAP2 coexpression clearly enhanced basal INa of ENaC that contained the mutant γ-ENaC with lysine at residue 138; moreover, subsequent trypsin exposure did not significantly add to the elevated basal INa (Fig. 11 B). These results indicate that ability of CAP2 coexpression to stimulate ENaC INa is exquisitely dependent on the residue at position 138 in γ-ENaC.


ENaC proteolytic regulation by channel-activating protease 2.

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

CAP2 stimulated α-R205A/R231A, β-, and γ-138K or α-, β-, and γ-138K plus 178–181 QQQQ but not α-R205A/R231A, β-, and γ-138A or α-, β-, and γ-138A plus 178–181 QQQQ mutant channels. (A) CAP2 induced INa of WT and α-R205A/R231A, β-, γ-, or α-R205A/R231A, β-, and γ-R138A or α-R205A/R231A, β-, and γ-R138K ENaC mutant channels (n = 12). (B) CAP2 induced INa of α-, β-, and γ-R138A, R178Q, K179Q, R180Q, and K181Q or α-, β-, and γ-R138K, R178Q, K179Q, R180Q, and K181Q 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig11: CAP2 stimulated α-R205A/R231A, β-, and γ-138K or α-, β-, and γ-138K plus 178–181 QQQQ but not α-R205A/R231A, β-, and γ-138A or α-, β-, and γ-138A plus 178–181 QQQQ mutant channels. (A) CAP2 induced INa of WT and α-R205A/R231A, β-, γ-, or α-R205A/R231A, β-, and γ-R138A or α-R205A/R231A, β-, and γ-R138K ENaC mutant channels (n = 12). (B) CAP2 induced INa of α-, β-, and γ-R138A, R178Q, K179Q, R180Q, and K181Q or α-, β-, and γ-R138K, R178Q, K179Q, R180Q, and K181Q 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.
Mentions: To further test the importance of γ-ENaC residue 138 in CAP2 stimulation of ENaC, we coexpressed either WT γ or γ-R138A or γ-R138K with α-R205A/R231A and WT β-ENaC. INa was stimulated by CAP2 coexpression only when an arginine (WT) or a lysine was present at position 138 in γ-ENaC (Fig. 11 A). We further isolated the action of CAP2 by coexpressing CAP2 with WT α- and β-ENaC combined with γ-178-181 QQQQ mutants that contained either alanine or lysine at γ-ENaC residue 138. These mutant γ-subunits are not expected to be cleaved by convertases acting at the γ-ENaC furin site, and they lack the polybasic tract implicated in CAP1 stimulation of mouse ENaC. CAP2 did not stimulate basal INa of ENaC with the double mutant γ-subunit containing alanine at residue 138; however, INa was markedly stimulated by subsequent trypsin (Fig. 11 B). In contrast, CAP2 coexpression clearly enhanced basal INa of ENaC that contained the mutant γ-ENaC with lysine at residue 138; moreover, subsequent trypsin exposure did not significantly add to the elevated basal INa (Fig. 11 B). These results indicate that ability of CAP2 coexpression to stimulate ENaC INa is exquisitely dependent on the residue at position 138 in γ-ENaC.

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