Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY motifs of the channel but is independent of sodium feedback inhibition

Abstract

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na+, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na+ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na+ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel's PY motifs, but it is not due to the interruption of Na+ feedback regulation.

(NDRG2) Stimulates Amiloride-sensitive Na+ Currents in Xenopus laevis Oocytes and Fisher Rat Thyroid Cells

“…1B). This confirms previous findings that the apparent intracellular Na ϩ concentration can be efficiently lowered in ENaC-expressing oocytes by maintaining the oocytes in a low Na ϩ bath solution (33,36,41). However, as shown in Fig.…”

“…Moreover, we determined ⌬I ami on 3 consecutive days after cRNA injection and incubated the oocytes in low Na ϩ to prevent Na ϩ overload and deterioration of the oocytes. As previously reported, the Sgk1 effect is preserved in oocytes maintained under these conditions (36). In the experiment shown in Fig.…”

“…To test this possibility we performed co-expression experiments in oocytes that were maintained in the presence of a low extracellular Na ϩ concentration after cRNA injection. This maneuver is known to lower the intracellular Na ϩ concentration of ENaC-expressing oocytes and to largely reduce sodium feedback inhibition (33,36,41). As expected, the reversal potential was shifted to the right in oocytes incubated in low extracellular Na ϩ ( Fig.…”

Regulation of voltage-gated ion channels in excitable cells by the ubiquitin ligases Nedd4 and Nedd4-2

“…53 In the normal kidney, ubiquitination by Nedd4-2 plays a key role in controlling the levels of ENaC at the luminal surface of the kidney tubules by targeting their degradation and thereby regulating Na + reabsorption. 54,55 In Liddle's syndrome, mutations in the C-terminal region of ENaC prevent this interaction with Nedd4-2. This prevents the degradation of ENaC which in turn leads to an accumulation of ENaC at the specificity and diversity of ubiquitin mediated protein regulation contributing to many cellular functions, such as the activation and silencing of transcription, signal transduction, apoptosis, immune and inflammatory response, cell cycle, receptor mediated endocytosis and various metabolic pathways such as autophagy.…”

“…55,56 The number of target proteins for Nedd4 and Nedd4-2 continues to grow and it is clear that Nedd4 and Nedd4-2 are two distinct proteins with separate physiological effectors. This is highlighted in a recent proteomic screen demonstrating that although they are closely related and share a similar structure, they have distinct substrates and thus may have distinct physiological functions.…”