The phosphorylation site T613 in the $\beta$-subunit of rat epithelial Na+ channel (ENaC) modulates channel inhibition by Nedd4-2

The epithelial Na+ channel (ENaC) is a heteromeric channel composed of three subunits ($\alpha$, $\beta$, $\gamma$). At the C-terminus of each subunit, a PY-motif allows binding of the ubiquitin ligase Nedd4-2 which plays a key role in promoting ENaC retrieval from the plasma membrane. Phosphorylation of Nedd4-2 by the serum and glucocorticoid-inducible kinase 1 (Sgk1) reduces Nedd4-2 binding to the PY-motifs. In $\beta$ and $\gamma$ENaC, threonine residues ($\beta$T613, $\gamma$T623) belong to an extracellular signal-regulated kinase (ERK) motif and directly precede the PY-motifs. Thus, phosphorylation of these residues may modulate the interaction of their adjacent PY-motifs with Nedd4-2. In this study, a phosphospecific antibody was used to demonstrate phosphorylation of $\beta$T613 in Xenopus laevis oocytes heterologously expressing rat $\alpha$$\beta$$\gamma$ENaC. Treating the oocytes with progesterone to stimulate ERK increased phosphorylation of $\beta$T613. Inactivation of the putative phosphorylation sites by mutating both threonine residues to alanine ($\beta$T613A/$\gamma$T623A) increased ENaC-mediated amiloride-sensitive whole-cell currents ($\Delta$Iami) and expression of $\beta$ENaC at the cell surface. Co-expression of Nedd4-2 largely reduced $\Delta$Iami in oocytes expressing $\alpha$$\beta$$\gamma$ENaC or channels with mutated PY-motifs in $\alpha$ and $\gamma$ENaC or in $\alpha$ and $\beta$ENaC. Importantly, the inhibitory effect of co-expressed Nedd4-2 was largely reduced in channels with mutated PY-motifs in $\alpha$ and $\gamma$ENaC when combined with the $\beta$T613A mutation but conserved in channels with mutated PY-motifs in $\alpha$ and $\beta$ENaC combined with the $\gamma$T623A mutation. These results suggest that phosphorylation and dephosphorylation of $\beta$T613 play a prominent role in regulating Nedd4-2-mediated ENaC retrieval from the plasma membrane.