Limits...
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.

Show MeSH
Representative strip-chart recordings of the short-circuit current, Isc, in (A) nontreated and (B) tetracycline-stimulated cells. The apical perfusion solution contained 10 μM CDPC except for the pulse periods when the blocker concentration was increased to 30 μM (arrows). The marked inhibition of current by 100 μM amiloride at the end of the experiments (arrowhead) confirmed that the Isc mostly reflects ENaC mediated Na+ currents. The amiloride sensitive currents are also subtracted from the short-circuit currents to determine macroscopic rates of sodium transport expressed as blocker-sensitive Na+ currents (INa).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233909&req=5

fig1: Representative strip-chart recordings of the short-circuit current, Isc, in (A) nontreated and (B) tetracycline-stimulated cells. The apical perfusion solution contained 10 μM CDPC except for the pulse periods when the blocker concentration was increased to 30 μM (arrows). The marked inhibition of current by 100 μM amiloride at the end of the experiments (arrowhead) confirmed that the Isc mostly reflects ENaC mediated Na+ currents. The amiloride sensitive currents are also subtracted from the short-circuit currents to determine macroscopic rates of sodium transport expressed as blocker-sensitive Na+ currents (INa).

Mentions: At the end of the experiments 100 μM amiloride was added to the apical perfusion solution to measure the amiloride-insensitive short-circuit sodium currents (see Fig. 1). These values were subtracted from the Isc measured previously to determine macroscopic rates of sodium transport expressed as INa.


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)

Representative strip-chart recordings of the short-circuit current, Isc, in (A) nontreated and (B) tetracycline-stimulated cells. The apical perfusion solution contained 10 μM CDPC except for the pulse periods when the blocker concentration was increased to 30 μM (arrows). The marked inhibition of current by 100 μM amiloride at the end of the experiments (arrowhead) confirmed that the Isc mostly reflects ENaC mediated Na+ currents. The amiloride sensitive currents are also subtracted from the short-circuit currents to determine macroscopic rates of sodium transport expressed as blocker-sensitive Na+ currents (INa).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233909&req=5

fig1: Representative strip-chart recordings of the short-circuit current, Isc, in (A) nontreated and (B) tetracycline-stimulated cells. The apical perfusion solution contained 10 μM CDPC except for the pulse periods when the blocker concentration was increased to 30 μM (arrows). The marked inhibition of current by 100 μM amiloride at the end of the experiments (arrowhead) confirmed that the Isc mostly reflects ENaC mediated Na+ currents. The amiloride sensitive currents are also subtracted from the short-circuit currents to determine macroscopic rates of sodium transport expressed as blocker-sensitive Na+ currents (INa).
Mentions: At the end of the experiments 100 μM amiloride was added to the apical perfusion solution to measure the amiloride-insensitive short-circuit sodium currents (see Fig. 1). These values were subtracted from the Isc measured previously to determine macroscopic rates of sodium transport expressed as INa.

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