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Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells.

Alvarez de la Rosa D, Li H, Canessa CM - J. Gen. Physiol. (2002)

Bottom Line: The biosynthesis of new channels can be followed by acquisition of endoglycosidase H-resistant oligosacharides in alpha and beta subunits and, in the case of alpha, by the appearance of a form resistant to reducing agents.Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits.Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The collecting duct regulates Na(+) transport by adjusting the abundance/activity of epithelial Na(+) channels (ENaC). In this study we have investigated the synthesis, degradation, endocytosis, and activity of ENaC and the effects of aldosterone on these processes using endogenous channels expressed in the A6 cell line. Biochemical studies were performed with a newly raised set of specific antibodies against each of the three subunits of the amphibian ENaC. Our results indicate simultaneous transcription and translation of alpha, beta, and gamma subunits and enhancement of both processes by aldosterone: two- and fourfold increase, respectively. The biosynthesis of new channels can be followed by acquisition of endoglycosidase H-resistant oligosacharides in alpha and beta subunits and, in the case of alpha, by the appearance of a form resistant to reducing agents. The half-life of the total pool of subunits (t(1/2) 40-70 min) is longer than the fraction of channels in the apical membrane (t(1/2) 12-17 min). Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits. Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.

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Antibody characterization and recognition of endogenously expressed ENaC in A6 cells. (A) A6 cells grown on plastic were transiently transfected with plasmid constructs containing α, β, or γ ENaC subunits. Cells were pulse-labeled for 15 min with [35S]-methionine and [35S]-cysteine and each subunit was immunoprecipitated with anti-Xenopus ENaC antibodies. Negative controls of mock-transfected cells were included in each experiment. (B) Polarized A6 cells grown on filters were [35S]-labeled for 2 h and endogenous ENaC subunits were immunoprecipitated with anti-Xenopus ENaC antibodies. Controls, including the immunogenic fusion proteins or with preimmune serums, are also shown. (C) Microsomal proteins from A6 cells grown on filters were resolved on a 10% SDS-PAGE, transferred to membranes, and detected with the specific xENaC antibodies by Western blots.
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fig1: Antibody characterization and recognition of endogenously expressed ENaC in A6 cells. (A) A6 cells grown on plastic were transiently transfected with plasmid constructs containing α, β, or γ ENaC subunits. Cells were pulse-labeled for 15 min with [35S]-methionine and [35S]-cysteine and each subunit was immunoprecipitated with anti-Xenopus ENaC antibodies. Negative controls of mock-transfected cells were included in each experiment. (B) Polarized A6 cells grown on filters were [35S]-labeled for 2 h and endogenous ENaC subunits were immunoprecipitated with anti-Xenopus ENaC antibodies. Controls, including the immunogenic fusion proteins or with preimmune serums, are also shown. (C) Microsomal proteins from A6 cells grown on filters were resolved on a 10% SDS-PAGE, transferred to membranes, and detected with the specific xENaC antibodies by Western blots.

Mentions: We developed specific antibodies against α, β, and γ subunits from xENaC. Specificity of the antibodies was first tested by their ability to immunoprecipitate xENaC subunits expressed in transiently transfected A6 cells and [35S]-radiolabeled for 15 min. α subunit was detected as a single band migrating at 85 kD, β subunit at 100 kD, and γ subunit at 90 kD (Fig. 1 A). Electrophoretic migration of each of the proteins corresponds to the molecular weight of the core glycosylated form as predicted by the sequence of the corresponding cDNA (Puoti et al., 1995). No signal was detected in untransfected cells (Fig. 1 A). We next used the antibodies to detect endogenous xENaC expressed in A6 cells grown on filters for at least 8 d and [35S]-radiolabeled for 2 h. The α antibody immunoprecipitated two bands, one at 85 kD, previously shown in transfected cells, and a second one that migrated at 65 kD. Both bands could be competed away by including GST-α fusion protein in the immunoprecipitation mix and were undetectable with preimmune serum (Fig. 1 B). Anti-β antibody immunoprecipitated proteins of 100 and 115 kD, whereas the γ antibody recovered a single band of 90 kD. In both cases the bands were not present when the immunoprecipitation was competed with the corresponding fusion protein or when preimmune serum was used (Fig. 1 B). Affinity-purified antibodies detected the same bands when used on Western blots of a crude microsomal fraction of A6 cells grown on filters (Fig. 1 C).


Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells.

Alvarez de la Rosa D, Li H, Canessa CM - J. Gen. Physiol. (2002)

Antibody characterization and recognition of endogenously expressed ENaC in A6 cells. (A) A6 cells grown on plastic were transiently transfected with plasmid constructs containing α, β, or γ ENaC subunits. Cells were pulse-labeled for 15 min with [35S]-methionine and [35S]-cysteine and each subunit was immunoprecipitated with anti-Xenopus ENaC antibodies. Negative controls of mock-transfected cells were included in each experiment. (B) Polarized A6 cells grown on filters were [35S]-labeled for 2 h and endogenous ENaC subunits were immunoprecipitated with anti-Xenopus ENaC antibodies. Controls, including the immunogenic fusion proteins or with preimmune serums, are also shown. (C) Microsomal proteins from A6 cells grown on filters were resolved on a 10% SDS-PAGE, transferred to membranes, and detected with the specific xENaC antibodies by Western blots.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233818&req=5

fig1: Antibody characterization and recognition of endogenously expressed ENaC in A6 cells. (A) A6 cells grown on plastic were transiently transfected with plasmid constructs containing α, β, or γ ENaC subunits. Cells were pulse-labeled for 15 min with [35S]-methionine and [35S]-cysteine and each subunit was immunoprecipitated with anti-Xenopus ENaC antibodies. Negative controls of mock-transfected cells were included in each experiment. (B) Polarized A6 cells grown on filters were [35S]-labeled for 2 h and endogenous ENaC subunits were immunoprecipitated with anti-Xenopus ENaC antibodies. Controls, including the immunogenic fusion proteins or with preimmune serums, are also shown. (C) Microsomal proteins from A6 cells grown on filters were resolved on a 10% SDS-PAGE, transferred to membranes, and detected with the specific xENaC antibodies by Western blots.
Mentions: We developed specific antibodies against α, β, and γ subunits from xENaC. Specificity of the antibodies was first tested by their ability to immunoprecipitate xENaC subunits expressed in transiently transfected A6 cells and [35S]-radiolabeled for 15 min. α subunit was detected as a single band migrating at 85 kD, β subunit at 100 kD, and γ subunit at 90 kD (Fig. 1 A). Electrophoretic migration of each of the proteins corresponds to the molecular weight of the core glycosylated form as predicted by the sequence of the corresponding cDNA (Puoti et al., 1995). No signal was detected in untransfected cells (Fig. 1 A). We next used the antibodies to detect endogenous xENaC expressed in A6 cells grown on filters for at least 8 d and [35S]-radiolabeled for 2 h. The α antibody immunoprecipitated two bands, one at 85 kD, previously shown in transfected cells, and a second one that migrated at 65 kD. Both bands could be competed away by including GST-α fusion protein in the immunoprecipitation mix and were undetectable with preimmune serum (Fig. 1 B). Anti-β antibody immunoprecipitated proteins of 100 and 115 kD, whereas the γ antibody recovered a single band of 90 kD. In both cases the bands were not present when the immunoprecipitation was competed with the corresponding fusion protein or when preimmune serum was used (Fig. 1 B). Affinity-purified antibodies detected the same bands when used on Western blots of a crude microsomal fraction of A6 cells grown on filters (Fig. 1 C).

Bottom Line: The biosynthesis of new channels can be followed by acquisition of endoglycosidase H-resistant oligosacharides in alpha and beta subunits and, in the case of alpha, by the appearance of a form resistant to reducing agents.Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits.Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The collecting duct regulates Na(+) transport by adjusting the abundance/activity of epithelial Na(+) channels (ENaC). In this study we have investigated the synthesis, degradation, endocytosis, and activity of ENaC and the effects of aldosterone on these processes using endogenous channels expressed in the A6 cell line. Biochemical studies were performed with a newly raised set of specific antibodies against each of the three subunits of the amphibian ENaC. Our results indicate simultaneous transcription and translation of alpha, beta, and gamma subunits and enhancement of both processes by aldosterone: two- and fourfold increase, respectively. The biosynthesis of new channels can be followed by acquisition of endoglycosidase H-resistant oligosacharides in alpha and beta subunits and, in the case of alpha, by the appearance of a form resistant to reducing agents. The half-life of the total pool of subunits (t(1/2) 40-70 min) is longer than the fraction of channels in the apical membrane (t(1/2) 12-17 min). Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits. Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.

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