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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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Amiloride-sensitive INa of triple mutant α-K561A, β-R503A, and γ-R515A channels activated by CAP2. WT α-, β-, γ-, or mutant α-K561A, β-R503A, and γ-R515A ENaC cRNA (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. 24 h after injection, two-electrode voltage clamp assays were conducted. Currents measured in the presence and absence of 10 μM amiloride, while clamping the membrane voltage to −100 mV, were digitized and recorded. Batches of oocytes were extracted from four different frogs (n = 24). Results are expressed as the means ± SE. Statistical significance was determined using an unpaired Student's t test. Basal WT INa versus WT trypsin-stimulated INa: *, P < 0.0001; basal WT INa versus basal triple mutant INa: **, P < 0.0011; basal triple mutant INa versus triple mutant trypsin-stimulated INa: #, significant P < 0.0001.
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fig6: Amiloride-sensitive INa of triple mutant α-K561A, β-R503A, and γ-R515A channels activated by CAP2. WT α-, β-, γ-, or mutant α-K561A, β-R503A, and γ-R515A ENaC cRNA (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. 24 h after injection, two-electrode voltage clamp assays were conducted. Currents measured in the presence and absence of 10 μM amiloride, while clamping the membrane voltage to −100 mV, were digitized and recorded. Batches of oocytes were extracted from four different frogs (n = 24). Results are expressed as the means ± SE. Statistical significance was determined using an unpaired Student's t test. Basal WT INa versus WT trypsin-stimulated INa: *, P < 0.0001; basal WT INa versus basal triple mutant INa: **, P < 0.0011; basal triple mutant INa versus triple mutant trypsin-stimulated INa: #, significant P < 0.0001.

Mentions: The highly conserved residue we found to be required for pre-M2 CAP2 fragments is 15 residues upstream of the degenerin site present in each subunit (Fig. 5 B). To assess the functional effect of this cleavage event in the pre-M2 region of the channel, we coexpressed a triple mutant channel (α-K561A, β-R503A, and γ-R515A) in oocytes with or without CAP2 and measured INa. We used trypsin to evaluate the proteolytic state of the WT or mutant channels, given previous reports by Caldwell et al. (2004) that trypsin activates near-silent channels in excised patches. Although the basal INa of the triple mutant channel was significantly diminished (751 ± 56 nA) compared with the basal INa of WT channels (1,652 ± 184 nA) (Fig. 6), subsequent trypsin exposure activated the INa of mutant channels (3.9-fold) and WT channels (3.9-fold) similarly. CAP2 coexpression increased basal INa of triple mutant channels by 3.43-fold, and trypsin had no further effect. In comparison, WT channels were activated by CAP2 (2.43-fold), and no further trypsin response was observed (Fig. 6). Thus, we identified a specific and highly conserved residue in the pre-M2 region of α-, β-, and γ-ENaC that is cleaved when the channels are expressed with CAP2. However, this cleavage event is not required for the stimulation of ENaC by coexpressed CAP2.


ENaC proteolytic regulation by channel-activating protease 2.

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

Amiloride-sensitive INa of triple mutant α-K561A, β-R503A, and γ-R515A channels activated by CAP2. WT α-, β-, γ-, or mutant α-K561A, β-R503A, and γ-R515A ENaC cRNA (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. 24 h after injection, two-electrode voltage clamp assays were conducted. Currents measured in the presence and absence of 10 μM amiloride, while clamping the membrane voltage to −100 mV, were digitized and recorded. Batches of oocytes were extracted from four different frogs (n = 24). Results are expressed as the means ± SE. Statistical significance was determined using an unpaired Student's t test. Basal WT INa versus WT trypsin-stimulated INa: *, P < 0.0001; basal WT INa versus basal triple mutant INa: **, P < 0.0011; basal triple mutant INa versus triple mutant trypsin-stimulated INa: #, significant P < 0.0001.
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fig6: Amiloride-sensitive INa of triple mutant α-K561A, β-R503A, and γ-R515A channels activated by CAP2. WT α-, β-, γ-, or mutant α-K561A, β-R503A, and γ-R515A ENaC cRNA (0.3 ng each) and 1 ng CAP2 cRNA were injected into oocytes. 24 h after injection, two-electrode voltage clamp assays were conducted. Currents measured in the presence and absence of 10 μM amiloride, while clamping the membrane voltage to −100 mV, were digitized and recorded. Batches of oocytes were extracted from four different frogs (n = 24). Results are expressed as the means ± SE. Statistical significance was determined using an unpaired Student's t test. Basal WT INa versus WT trypsin-stimulated INa: *, P < 0.0001; basal WT INa versus basal triple mutant INa: **, P < 0.0011; basal triple mutant INa versus triple mutant trypsin-stimulated INa: #, significant P < 0.0001.
Mentions: The highly conserved residue we found to be required for pre-M2 CAP2 fragments is 15 residues upstream of the degenerin site present in each subunit (Fig. 5 B). To assess the functional effect of this cleavage event in the pre-M2 region of the channel, we coexpressed a triple mutant channel (α-K561A, β-R503A, and γ-R515A) in oocytes with or without CAP2 and measured INa. We used trypsin to evaluate the proteolytic state of the WT or mutant channels, given previous reports by Caldwell et al. (2004) that trypsin activates near-silent channels in excised patches. Although the basal INa of the triple mutant channel was significantly diminished (751 ± 56 nA) compared with the basal INa of WT channels (1,652 ± 184 nA) (Fig. 6), subsequent trypsin exposure activated the INa of mutant channels (3.9-fold) and WT channels (3.9-fold) similarly. CAP2 coexpression increased basal INa of triple mutant channels by 3.43-fold, and trypsin had no further effect. In comparison, WT channels were activated by CAP2 (2.43-fold), and no further trypsin response was observed (Fig. 6). Thus, we identified a specific and highly conserved residue in the pre-M2 region of α-, β-, and γ-ENaC that is cleaved when the channels are expressed with CAP2. However, this cleavage event is not required for the stimulation of ENaC by coexpressed CAP2.

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