# Acute activation of human epithelial sodium channel (ENaC) by serum and glucocorticoid inducible kinase 1 (SGK1) requires prior cleavage of the channel’s γ-subunit at its proximal cleavage site

## TL;DR

This study shows that SGK1 activates the human ENaC sodium channel only after a specific cleavage in the γ-subunit.

## Contribution

The study reveals a novel requirement for prior γ-subunit cleavage for SGK1-mediated activation of human ENaC.

## Key findings

- Human ENaC can be acutely activated by SGK1 at the homologous phosphorylation site S594.
- SGK1 activation of ENaC depends on prior cleavage of the γ-subunit at its proximal cleavage site.
- Tethering the inhibitory peptide in γENaC prevents SGK1 stimulation of the channel.

## Abstract

Serum and glucocorticoid inducible kinase 1 (SGK1) is a key regulator of the epithelial sodium channel (ENaC). In rat ENaC, the serine residue 621 (S621) in the channel’s α-subunit is essential for acute channel activation by SGK1 in outside-out patches. Phosphorylation at S621 probably turns previously silent channels into channels with a high open probability. This is reminiscent of proteolytic ENaC activation resulting from cleavage of the channel’s γ-subunit at specific proximal and distal cleavage sites and the release of an inhibitory peptide tract. The first aim of this study was to demonstrate that human ENaC could also be activated acutely by SGK1 and that this depended on the homologous phosphorylation site S594 in human αENaC. Secondly, we wanted to explore whether human ENaC activation by SGK1 depended on the cleavage state of γENaC. Outside-out patch-clamp recordings in Xenopus laevis oocytes expressing human αβγENaC revealed the critical importance of S594 for acute channel activation by SGK1. The latter was not additive to proteolytic channel activation. Interestingly, preventing proximal cleavage in human γENaC completely abolished the stimulatory effect of SGK1. Moreover, tethering the inhibitory peptide in γENaC to its binding site via an engineered disulfide bond prevented stimulation by SGK1. We conclude that ENaC activation by SGK1 requires prior cleavage of γENaC at its proximal cleavage site. Together, these results reveal that SGK1-mediated stimulation of human ENaC is intricately linked to the proteolytic processing of the channel’s γ-subunit, emphasizing a previously underappreciated interplay between kinase and protease regulatory pathways.

The online version contains supplementary material available at 10.1007/s00424-025-03099-z.