Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

Abstract

The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with 35 S or 32 P. When immune complexes were resolved on SDS-PAGE, the ␣-subunit migrated at 85 and 65 kDa, the ␤-subunit at 115 and 100 kDa, and the ␥-subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The ␣-subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of ␣ is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of ␣-, ␤-, and ␥-subunits approximately twofold. Exposure to 1 M aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport. epithelial sodium channel; aldosterone; sodium transport IT IS NOW WIDELY ACCEPTED that the epithelial sodium channel (ENaC), located in the cortical collecting duct of the renal tubule, plays a major role in preserving sodium balance (9). Although substantial progress has been made in understanding ENaC regulation at the molecular level, there are fewer insights into the cascade of molecular events that link activation of cell membrane receptors or intracellular receptors, such as the mineralocorticoid receptor, to the activation of ENaC located in the apical membrane of epithelial cells.In this study we sought to examine the question whether phosphorylation of one or more of the three subunits that comprise ENaC is involved in regulation of the sodium channel. Our interest was prompted, at least in part, by the previous study of Shimkets et al. (14), who explored MDCK cells that were transfected with cDNAs of all three rat ENaC subunits. After separation of the subunits by immunoprecipitation, phosphorylation was found in the ␤-and ␥-subunits, but not in the ␣-subunit, in resting cells. Furthermore, the administration of aldosterone or insulin markedly increased phosphorylation of ␤-and ␥-subunits by the transfer of phosphate to residues located in the carboxy terminus (14). If these observations were confirmed in cells with endogenous ENaC and genetic regulation of signals that control sodium channe...

The T8590C Polymorphism of CYP4A11 and 20-Hydroxyeicosatetraenoic Acid in Essential Hypertension

“…It is conceivable that inhibition of outer renal medullary CYP4A isoforms is a mechanism by which insulin increases sodium reabsorption at the thick ascending limb, because insulin also exerts this action at the proximal tubule by translational and posttranslational activation of the NHE3 isoform of the Na ϩ /H ϩ exchanger 30 ; at the thiazide-sensitive Na ϩ Cl Ϫ transporter via diminished expression of WNK4 kinase 31 ; and at the epithelial sodium channel, via phosphorylation and translocation of the 3 channel subunits to the membrane. 32 This publication extends our previous observations in 2 regards. First, whereas Dahl-S rats are an inbred strain in which salt sensitivity of blood pressure coexists with insulin resistance, 18 salt-sensitive hypertensive subjects are not always insulin resistant.…”

Insulin's impact on renal sodium transport and blood pressure in health, obesity, and diabetes

“…Furthermore, insulin may acutely alter ENaC activity by mechanisms in addition to trafficking, perhaps by changing open probability or activity of existing channels. For example, Canessa and associates (136,169) have demonstrated that insulin phosphorylates ENaC subunits in both transfected Madin-Darby canine kidney cells and endogenously ENaC-expressing A6 cells. In A6 cells, this phosphorylation occurred on all three subunits and was associated with increased activity (169).…”

“…For example, Canessa and associates (136,169) have demonstrated that insulin phosphorylates ENaC subunits in both transfected Madin-Darby canine kidney cells and endogenously ENaC-expressing A6 cells. In A6 cells, this phosphorylation occurred on all three subunits and was associated with increased activity (169). Figure 2 illustrates how insulin might increase sodium reabsorption in the CD principal cell by activating both ENaC and Na-K-ATPase.…”

The Cyp2c44 Epoxygenase Regulates Epithelial Sodium Channel Activity and the Blood Pressure Responses to Increased Dietary Salt

“…Thus, Liddle syndrome, a form of severe salt-sensitive hypertension, is caused by gain of function mutations/truncations in the channel ␤ or ␥ subunits (3,10,11), whereas loss of function mutations are associated with the hypotensive phenotype of pseudohypoaldosteronism type 1 (3,10,12). ENaC gating is regulated by hormones such as aldosterone, insulin, and EGF (13)(14)(15)(16). EGF inhibits ENaC (15,16) by mechanisms that involve an ERK1/2-catalyzed threonine phosphorylation of ENaC␤ and ENaC␥ (17,18).…”