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Mechanisms of regulation of epithelial sodium channel by SGK1 in A6 cells.

Alvarez de la Rosa D, Paunescu TG, Els WJ, Helman SI, Canessa CM - J. Gen. Physiol. (2004)

Bottom Line: Using noise analysis we demonstrate that SGK1 effect on Isc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability.SGK1T(S425D) also produced a 1.6-1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane.We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

View Article: PubMed Central - PubMed

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

ABSTRACT
The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1T(S425D)) induced a sixfold increase in amiloride-sensitive short-circuit current (Isc). Using noise analysis we demonstrate that SGK1 effect on Isc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1T(S425D) increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1T(S425D) also produced a 1.6-1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

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Quantification of total and plasma membrane ENaC in A6 cells. We used cell surface biotinylation to obtain an estimate of the number of ENaCs endogenously expressed in the plasma membrane of A6 cells. (A) Coomassie blue staining of a SDS-PAGE with the product of MBP–αENaC fusion protein purification. Molecular mass (kD) of each of the standards is indicated on the left. (B) A serial dilution of the MBP–αENaC fusion protein was detected by Western blot with a polyclonal antibody against α subunit. Signal intensities were quantified with a densitometer and plotted against the amount of fusion protein in fmol. The signal was linear between 5 and 20 fmol. (C) Samples from total protein (T) and biotinylated plasma membrane proteins (PM) were loaded in the same gel used for the serial dilution of fusion protein.
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fig7: Quantification of total and plasma membrane ENaC in A6 cells. We used cell surface biotinylation to obtain an estimate of the number of ENaCs endogenously expressed in the plasma membrane of A6 cells. (A) Coomassie blue staining of a SDS-PAGE with the product of MBP–αENaC fusion protein purification. Molecular mass (kD) of each of the standards is indicated on the left. (B) A serial dilution of the MBP–αENaC fusion protein was detected by Western blot with a polyclonal antibody against α subunit. Signal intensities were quantified with a densitometer and plotted against the amount of fusion protein in fmol. The signal was linear between 5 and 20 fmol. (C) Samples from total protein (T) and biotinylated plasma membrane proteins (PM) were loaded in the same gel used for the serial dilution of fusion protein.

Mentions: The previous results suggest that not all channels in the apical membrane participate in sodium transport but there is a pool of inactive channels that can be activated by SGK1TS425D. To test this hypothesis we estimated the total number of xENaC α subunit in the plasma membrane using a quantitative biotinylation approach. A standard curve was constructed with a purified MBP fusion protein containing αENaC sequence used to raise the antibody loaded in decreasing amounts on a SDS-PAGE gel and analyzed by Western blotting with anti-αENaC antibody (Fig. 7, A and B). Signal intensities measured by densitometry were linear between 5 and 20 fmol and were used to construct the standard curve (Fig. 7 B). Samples of total protein and plasma membrane protein recovered by biotinylation from A6 cells grown on filters in control conditions were loaded in parallel lanes of the same Western blot (Fig. 7 C). By comparison with the standard curve we estimated that the content of α subunit was 15.8 fmol in the total protein sample and 6.2 fmol in the cell-surface protein sample. These values allowed to calculate the total amounts recovered from the original 4.7-cm2 filters, which was 455 μg. Assuming a cell surface area of 100 μm2, the filter contained 4.7 × 106 cells. Thus, the total protein sample loaded on the gel (19.5 μg) corresponds approximately to 0.29 × 106 cells and the cell-surface protein sample (61 μg) corresponds to 0.63 × 106 cells. This gives ∼4.7 × 104 molecules of α subunit per cell and 0.6 × 104 molecules in the membrane. If all molecules are forming channels and the stoichiometry of ENaC is 2α:1β:1γ (Firsov et al., 1998), each cell would have a total of 2.35 × 104 channels, 0.3 × 104 of them located in the plasma membrane. In other words, ∼13% of all the α subunits is expressed in the plasma membrane. This number is in agreement with previous results obtained with the A6 parental cell line (Weisz et al., 2000), where it was calculated that ∼20% of each of the subunits is in the plasma membrane. Thus, using a biochemical method to estimate the number of channels in the plasma membrane, we obtained a value that is approximately two orders of magnitude higher than the number of functional channels revealed by blocker-induced noise analysis. Nonetheless, it is important to highlight that this method involves many experimental steps that introduce errors in the quantification, as well as several assumptions that together render the final value somewhat inaccurate. We could not confirm our conclusions with data from the β and γ subunits because of difficulties in the purification of the MBP fusion proteins. Therefore, the results of our calculation should be regarded as a gross estimate rather than a precise value.


Mechanisms of regulation of epithelial sodium channel by SGK1 in A6 cells.

Alvarez de la Rosa D, Paunescu TG, Els WJ, Helman SI, Canessa CM - J. Gen. Physiol. (2004)

Quantification of total and plasma membrane ENaC in A6 cells. We used cell surface biotinylation to obtain an estimate of the number of ENaCs endogenously expressed in the plasma membrane of A6 cells. (A) Coomassie blue staining of a SDS-PAGE with the product of MBP–αENaC fusion protein purification. Molecular mass (kD) of each of the standards is indicated on the left. (B) A serial dilution of the MBP–αENaC fusion protein was detected by Western blot with a polyclonal antibody against α subunit. Signal intensities were quantified with a densitometer and plotted against the amount of fusion protein in fmol. The signal was linear between 5 and 20 fmol. (C) Samples from total protein (T) and biotinylated plasma membrane proteins (PM) were loaded in the same gel used for the serial dilution of fusion protein.
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Related In: Results  -  Collection

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fig7: Quantification of total and plasma membrane ENaC in A6 cells. We used cell surface biotinylation to obtain an estimate of the number of ENaCs endogenously expressed in the plasma membrane of A6 cells. (A) Coomassie blue staining of a SDS-PAGE with the product of MBP–αENaC fusion protein purification. Molecular mass (kD) of each of the standards is indicated on the left. (B) A serial dilution of the MBP–αENaC fusion protein was detected by Western blot with a polyclonal antibody against α subunit. Signal intensities were quantified with a densitometer and plotted against the amount of fusion protein in fmol. The signal was linear between 5 and 20 fmol. (C) Samples from total protein (T) and biotinylated plasma membrane proteins (PM) were loaded in the same gel used for the serial dilution of fusion protein.
Mentions: The previous results suggest that not all channels in the apical membrane participate in sodium transport but there is a pool of inactive channels that can be activated by SGK1TS425D. To test this hypothesis we estimated the total number of xENaC α subunit in the plasma membrane using a quantitative biotinylation approach. A standard curve was constructed with a purified MBP fusion protein containing αENaC sequence used to raise the antibody loaded in decreasing amounts on a SDS-PAGE gel and analyzed by Western blotting with anti-αENaC antibody (Fig. 7, A and B). Signal intensities measured by densitometry were linear between 5 and 20 fmol and were used to construct the standard curve (Fig. 7 B). Samples of total protein and plasma membrane protein recovered by biotinylation from A6 cells grown on filters in control conditions were loaded in parallel lanes of the same Western blot (Fig. 7 C). By comparison with the standard curve we estimated that the content of α subunit was 15.8 fmol in the total protein sample and 6.2 fmol in the cell-surface protein sample. These values allowed to calculate the total amounts recovered from the original 4.7-cm2 filters, which was 455 μg. Assuming a cell surface area of 100 μm2, the filter contained 4.7 × 106 cells. Thus, the total protein sample loaded on the gel (19.5 μg) corresponds approximately to 0.29 × 106 cells and the cell-surface protein sample (61 μg) corresponds to 0.63 × 106 cells. This gives ∼4.7 × 104 molecules of α subunit per cell and 0.6 × 104 molecules in the membrane. If all molecules are forming channels and the stoichiometry of ENaC is 2α:1β:1γ (Firsov et al., 1998), each cell would have a total of 2.35 × 104 channels, 0.3 × 104 of them located in the plasma membrane. In other words, ∼13% of all the α subunits is expressed in the plasma membrane. This number is in agreement with previous results obtained with the A6 parental cell line (Weisz et al., 2000), where it was calculated that ∼20% of each of the subunits is in the plasma membrane. Thus, using a biochemical method to estimate the number of channels in the plasma membrane, we obtained a value that is approximately two orders of magnitude higher than the number of functional channels revealed by blocker-induced noise analysis. Nonetheless, it is important to highlight that this method involves many experimental steps that introduce errors in the quantification, as well as several assumptions that together render the final value somewhat inaccurate. We could not confirm our conclusions with data from the β and γ subunits because of difficulties in the purification of the MBP fusion proteins. Therefore, the results of our calculation should be regarded as a gross estimate rather than a precise value.

Bottom Line: Using noise analysis we demonstrate that SGK1 effect on Isc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability.SGK1T(S425D) also produced a 1.6-1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane.We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

View Article: PubMed Central - PubMed

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

ABSTRACT
The serum and glucocorticoid induced kinase 1 (SGK1) participates in the regulation of sodium reabsorption in the distal segment of the renal tubule, where it may modify the function of the epithelial sodium channel (ENaC). The molecular mechanism underlying SGK1 regulation of ENaC in renal epithelial cells remains controversial. We have addressed this issue in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible system. Expression of a constitutively active mutant of SGK1 (SGK1T(S425D)) induced a sixfold increase in amiloride-sensitive short-circuit current (Isc). Using noise analysis we demonstrate that SGK1 effect on Isc is due to a fourfold increase in the number of functional ENaCs in the membrane and a 43% increase in channel open probability. Impedance analysis indicated that SGK1T(S425D) increased the absolute value of cell equivalent capacitance by an average of 13.7%. SGK1T(S425D) also produced a 1.6-1.9-fold increase in total and plasma membrane subunit abundance, without changing the half-life of channels in the membrane. We conclude that in contrast to aldosterone, where stimulation of transport can be explained simply by an increase in channel synthesis, SGK1 effects are more complex and involve at least three actions: (1) increase of ENaC open probability; (2) increase of subunit abundance within apical membranes and intracellular compartments; and (3) activation of one or more pools of preexistent channels within the apical membranes and/or intracellular compartments.

Show MeSH