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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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Turnover of the total cellular pool of ENaC subunits without and with aldosterone. (A) Control cells or cells pretreated with aldosterone for 6 h were pulse-labeled with [35S]-methionine and [35S]-cysteine for 30 min and then chased with cold medium for the indicated periods of time (minutes). α, β, and γ subunits were immunoprecipitated from the lysates and resolved on SDS-PAGE. A representative autoradiograph of each subunit is shown. (B) Scanning densitometry values from autoradiographs shown in A. Values were normalized to the 0 time-point.
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fig6: Turnover of the total cellular pool of ENaC subunits without and with aldosterone. (A) Control cells or cells pretreated with aldosterone for 6 h were pulse-labeled with [35S]-methionine and [35S]-cysteine for 30 min and then chased with cold medium for the indicated periods of time (minutes). α, β, and γ subunits were immunoprecipitated from the lysates and resolved on SDS-PAGE. A representative autoradiograph of each subunit is shown. (B) Scanning densitometry values from autoradiographs shown in A. Values were normalized to the 0 time-point.

Mentions: We next investigated the possibility that phosphorylation could account for the aberrant migration in SDS-PAGE. Immunoprecipitated [35S]-radiolabeled αxENaC was treated with phosphatases (calf intestinal alkaline phosphatase or shrimp alkaline phosphatase). Neither of these enzymes altered the migration of the two bands, indicating that phosphorylation is not implicated in the change of mobility (Fig. 3 B). In contrast, alkylation with iodoacetamide (IAA) in the presence of the reducing agent DTT completely eliminated the lower band (Fig. 3 B). This result indicates that the Xenopus α subunit is prone to form disulfide bridges resistant to reducing agents. Moreover, the formation of stable disulfide bridges does not occur in recently synthesized protein, but after it has acquired resistance to Endo-H (Fig. 2). The most convincing evidence that the 65-kD band corresponds to a modification of the α subunit is provided by pulse-chase experiments (see Fig. 6 A), which will be described later in this section.


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)

Turnover of the total cellular pool of ENaC subunits without and with aldosterone. (A) Control cells or cells pretreated with aldosterone for 6 h were pulse-labeled with [35S]-methionine and [35S]-cysteine for 30 min and then chased with cold medium for the indicated periods of time (minutes). α, β, and γ subunits were immunoprecipitated from the lysates and resolved on SDS-PAGE. A representative autoradiograph of each subunit is shown. (B) Scanning densitometry values from autoradiographs shown in A. Values were normalized to the 0 time-point.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Turnover of the total cellular pool of ENaC subunits without and with aldosterone. (A) Control cells or cells pretreated with aldosterone for 6 h were pulse-labeled with [35S]-methionine and [35S]-cysteine for 30 min and then chased with cold medium for the indicated periods of time (minutes). α, β, and γ subunits were immunoprecipitated from the lysates and resolved on SDS-PAGE. A representative autoradiograph of each subunit is shown. (B) Scanning densitometry values from autoradiographs shown in A. Values were normalized to the 0 time-point.
Mentions: We next investigated the possibility that phosphorylation could account for the aberrant migration in SDS-PAGE. Immunoprecipitated [35S]-radiolabeled αxENaC was treated with phosphatases (calf intestinal alkaline phosphatase or shrimp alkaline phosphatase). Neither of these enzymes altered the migration of the two bands, indicating that phosphorylation is not implicated in the change of mobility (Fig. 3 B). In contrast, alkylation with iodoacetamide (IAA) in the presence of the reducing agent DTT completely eliminated the lower band (Fig. 3 B). This result indicates that the Xenopus α subunit is prone to form disulfide bridges resistant to reducing agents. Moreover, the formation of stable disulfide bridges does not occur in recently synthesized protein, but after it has acquired resistance to Endo-H (Fig. 2). The most convincing evidence that the 65-kD band corresponds to a modification of the α subunit is provided by pulse-chase experiments (see Fig. 6 A), which will be described later in this section.

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.

Show MeSH