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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-ENaC K149A/R164A/K169A/R177A mutant on CAP2-induced INa and α-ENaC N-terminal fragments. (A) Western blots of α-ENaC N-terminal 17- and 19-kD novel fragments at the surface (top) and total pools (bottom). Lane 1, uninjected eggs; lane 2, ENaC alone; lane 3, ENaC plus CAP2; lane 4, α-ENaC mutant alone; lane 5, α-ENaC mutant plus CAP2. FL, full-length. A representative experiment is shown (n = 3). (B) INa of WT and mutant α-ENaC stimulated by CAP2. Batches of oocytes were extracted from three different frogs (n = 18). * and **, P < 0.0001. Amiloride-sensitive currents were measured as described in Fig. 1.
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fig4: Effect of α-ENaC K149A/R164A/K169A/R177A mutant on CAP2-induced INa and α-ENaC N-terminal fragments. (A) Western blots of α-ENaC N-terminal 17- and 19-kD novel fragments at the surface (top) and total pools (bottom). Lane 1, uninjected eggs; lane 2, ENaC alone; lane 3, ENaC plus CAP2; lane 4, α-ENaC mutant alone; lane 5, α-ENaC mutant plus CAP2. FL, full-length. A representative experiment is shown (n = 3). (B) INa of WT and mutant α-ENaC stimulated by CAP2. Batches of oocytes were extracted from three different frogs (n = 18). * and **, P < 0.0001. Amiloride-sensitive currents were measured as described in Fig. 1.

Mentions: CAP2 coexpression decreased the amount of full-length protein corresponding to all three ENaC subunits in whole cell lysates (Fig. 3). The loss of full-length ENaC coincided with the somewhat variable appearance of smaller fragments. CAP2 coexpression consistently enhanced ∼32-kD HA-NT and complementary ∼66-kD V5-CT staining bands of α-ENaC, suggesting increased cleavage at the furin-consensus sites (Fig. 3, A and B). In some experiments, CAP2 coexpression with α-ENaC generated an HA-NT band of ∼82 kD (Fig. 3 A), and more consistently yielded an ∼17-kD V5-CT band (Fig. 3 B). These complementary α-ENaC fragments predict cleavage close to the second transmembrane, or pre-M2, region. The relationship between CAP2-stimulated cleavage at the α-ENaC furin and pre-M2 regions to CAP2-stimulated INa is addressed in detail below. In addition, unique HA-NT fragments of α-ENaC of ∼17 and ∼19 kD were induced by CAP2, consistent with cleavage just distal of the first transmembrane segment (Fig. 3 A). Mutagenesis of four conserved basic residues in this region to alanine (K149A/R164A/K169A/R177A) prevented ∼17 and ∼19 kD N-terminal fragments with CAP2 coexpression, but it did not affect stimulation of INa (Fig. 4). Thus, we conclude that CAP2-mediated cleavage in the proximal region of the extracellular loop of the α-ENaC subunit is not required for CAP2 to stimulate ENaC.


ENaC proteolytic regulation by channel-activating protease 2.

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

Effect of α-ENaC K149A/R164A/K169A/R177A mutant on CAP2-induced INa and α-ENaC N-terminal fragments. (A) Western blots of α-ENaC N-terminal 17- and 19-kD novel fragments at the surface (top) and total pools (bottom). Lane 1, uninjected eggs; lane 2, ENaC alone; lane 3, ENaC plus CAP2; lane 4, α-ENaC mutant alone; lane 5, α-ENaC mutant plus CAP2. FL, full-length. A representative experiment is shown (n = 3). (B) INa of WT and mutant α-ENaC stimulated by CAP2. Batches of oocytes were extracted from three different frogs (n = 18). * and **, P < 0.0001. Amiloride-sensitive currents were measured as described in Fig. 1.
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fig4: Effect of α-ENaC K149A/R164A/K169A/R177A mutant on CAP2-induced INa and α-ENaC N-terminal fragments. (A) Western blots of α-ENaC N-terminal 17- and 19-kD novel fragments at the surface (top) and total pools (bottom). Lane 1, uninjected eggs; lane 2, ENaC alone; lane 3, ENaC plus CAP2; lane 4, α-ENaC mutant alone; lane 5, α-ENaC mutant plus CAP2. FL, full-length. A representative experiment is shown (n = 3). (B) INa of WT and mutant α-ENaC stimulated by CAP2. Batches of oocytes were extracted from three different frogs (n = 18). * and **, P < 0.0001. Amiloride-sensitive currents were measured as described in Fig. 1.
Mentions: CAP2 coexpression decreased the amount of full-length protein corresponding to all three ENaC subunits in whole cell lysates (Fig. 3). The loss of full-length ENaC coincided with the somewhat variable appearance of smaller fragments. CAP2 coexpression consistently enhanced ∼32-kD HA-NT and complementary ∼66-kD V5-CT staining bands of α-ENaC, suggesting increased cleavage at the furin-consensus sites (Fig. 3, A and B). In some experiments, CAP2 coexpression with α-ENaC generated an HA-NT band of ∼82 kD (Fig. 3 A), and more consistently yielded an ∼17-kD V5-CT band (Fig. 3 B). These complementary α-ENaC fragments predict cleavage close to the second transmembrane, or pre-M2, region. The relationship between CAP2-stimulated cleavage at the α-ENaC furin and pre-M2 regions to CAP2-stimulated INa is addressed in detail below. In addition, unique HA-NT fragments of α-ENaC of ∼17 and ∼19 kD were induced by CAP2, consistent with cleavage just distal of the first transmembrane segment (Fig. 3 A). Mutagenesis of four conserved basic residues in this region to alanine (K149A/R164A/K169A/R177A) prevented ∼17 and ∼19 kD N-terminal fragments with CAP2 coexpression, but it did not affect stimulation of INa (Fig. 4). Thus, we conclude that CAP2-mediated cleavage in the proximal region of the extracellular loop of the α-ENaC subunit is not required for CAP2 to stimulate 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