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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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CAP2 and trypsin effects on amiloride-sensitive INa of WT or HA-NT– and V5-CT–tagged or β-S518C mutant channels in oocytes. WT α-, β-, γ-, or double tagged (HA-NT/V5-CT) and mutant α-, β-S518C, and γ-cRNA (0.3 ng each) ENaC subunits 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. (A) Stimulation of ENaC WT channels by coexpression of CAP2 and 2 μg/ml of exogenous trypsin (n = 24). (B) CAP2 and trypsin (2 μg/ml) stimulation of ENaC α-, β-, and γ-HA-NT/V5-CT–tagged subunits (n = 18). (C) 1 mM MTSET activation of α-, β-S518C, and γ mutant untagged channels with or without CAP2 (n = 24). (A–C) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. * and **, P < 0.0001, significant difference when CAP2- or trypsin-stimulated INa is compared with control basal INa. Statistical significance was determined using an unpaired Student's t test.
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fig1: CAP2 and trypsin effects on amiloride-sensitive INa of WT or HA-NT– and V5-CT–tagged or β-S518C mutant channels in oocytes. WT α-, β-, γ-, or double tagged (HA-NT/V5-CT) and mutant α-, β-S518C, and γ-cRNA (0.3 ng each) ENaC subunits 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. (A) Stimulation of ENaC WT channels by coexpression of CAP2 and 2 μg/ml of exogenous trypsin (n = 24). (B) CAP2 and trypsin (2 μg/ml) stimulation of ENaC α-, β-, and γ-HA-NT/V5-CT–tagged subunits (n = 18). (C) 1 mM MTSET activation of α-, β-S518C, and γ mutant untagged channels with or without CAP2 (n = 24). (A–C) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. * and **, P < 0.0001, significant difference when CAP2- or trypsin-stimulated INa is compared with control basal INa. Statistical significance was determined using an unpaired Student's t test.

Mentions: We applied functional and biochemical approaches in parallel to assess the effects of CAP2 on the activity, number, and cleavage of coexpressed ENaC in Xenopus oocytes. To establish that our experimental conditions were informative for these analyses, we first observed that coexpression of CAP2 with WT ENaC elevated basal INa by ∼2.5-fold over WT ENaC alone (Fig. 1 A). Whereas the basal INa of WT ENaC alone was stimulated ∼3.5-fold by the application of trypsin, the already elevated INa of oocytes coexpressing CAP2 and ENaC was not stimulated further by exogenous trypsin. We routinely observed a reduction in INa after 5 min of trypsin exposure of oocytes coexpressing ENaC and CAP2 (Fig. 1, A and B). This apparent inhibitory effect of trypsin is mainly due to rundown of the CAP2-stimulated channels during the interval of trypsin application.


ENaC proteolytic regulation by channel-activating protease 2.

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

CAP2 and trypsin effects on amiloride-sensitive INa of WT or HA-NT– and V5-CT–tagged or β-S518C mutant channels in oocytes. WT α-, β-, γ-, or double tagged (HA-NT/V5-CT) and mutant α-, β-S518C, and γ-cRNA (0.3 ng each) ENaC subunits 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. (A) Stimulation of ENaC WT channels by coexpression of CAP2 and 2 μg/ml of exogenous trypsin (n = 24). (B) CAP2 and trypsin (2 μg/ml) stimulation of ENaC α-, β-, and γ-HA-NT/V5-CT–tagged subunits (n = 18). (C) 1 mM MTSET activation of α-, β-S518C, and γ mutant untagged channels with or without CAP2 (n = 24). (A–C) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. * and **, P < 0.0001, significant difference when CAP2- or trypsin-stimulated INa is compared with control basal INa. Statistical significance was determined using an unpaired Student's t test.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: CAP2 and trypsin effects on amiloride-sensitive INa of WT or HA-NT– and V5-CT–tagged or β-S518C mutant channels in oocytes. WT α-, β-, γ-, or double tagged (HA-NT/V5-CT) and mutant α-, β-S518C, and γ-cRNA (0.3 ng each) ENaC subunits 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. (A) Stimulation of ENaC WT channels by coexpression of CAP2 and 2 μg/ml of exogenous trypsin (n = 24). (B) CAP2 and trypsin (2 μg/ml) stimulation of ENaC α-, β-, and γ-HA-NT/V5-CT–tagged subunits (n = 18). (C) 1 mM MTSET activation of α-, β-S518C, and γ mutant untagged channels with or without CAP2 (n = 24). (A–C) Batches of oocytes were extracted from four to five different frogs. Results are expressed as the means ± SE. * and **, P < 0.0001, significant difference when CAP2- or trypsin-stimulated INa is compared with control basal INa. Statistical significance was determined using an unpaired Student's t test.
Mentions: We applied functional and biochemical approaches in parallel to assess the effects of CAP2 on the activity, number, and cleavage of coexpressed ENaC in Xenopus oocytes. To establish that our experimental conditions were informative for these analyses, we first observed that coexpression of CAP2 with WT ENaC elevated basal INa by ∼2.5-fold over WT ENaC alone (Fig. 1 A). Whereas the basal INa of WT ENaC alone was stimulated ∼3.5-fold by the application of trypsin, the already elevated INa of oocytes coexpressing CAP2 and ENaC was not stimulated further by exogenous trypsin. We routinely observed a reduction in INa after 5 min of trypsin exposure of oocytes coexpressing ENaC and CAP2 (Fig. 1, A and B). This apparent inhibitory effect of trypsin is mainly due to rundown of the CAP2-stimulated channels during the interval of trypsin application.

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