Regulation of epithelial Na⁺ channel activity by conserved serine/threonine switches within sorting signals

The PY and YXXφ motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the μ₂-subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na⁺ channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)φ). Just preceding the PY and embedded within the YXXφ motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr⁶²⁸ preceding the PY motif in γ-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of γ-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXXφ motif of γ-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in γ-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr⁶²⁸ and Thr⁶³⁵ also disrupted ENaC precipitation with the μ₂-subunit of the AP-2 complex. Moreover, the YXXφ motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXXφ motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.