Identification of functional PI(4,5)P 2 and PI(3,4,5)P 3 binding sites in ENaC subunits

Phosphatidylinositides, including PI(4,5)P 2 and PI(3,4,5)P 3 , are signaling molecules that can modulate activity of the epithelial Na + channel (ENaC). Functional phospholipid binding sites within ENaC have not been fully defined. Here, we identify regions just following the second transmembrane domains in β‐ and γ‐ENaC important to PI(3,4,5)P 3 regulation. A similar region in α‐ENaC does not play a role in regulation of the channel by phospholipids, although, it affects gating independent of phospholipid binding. Importantly, we were able to firmly establish the regions in β‐ and γ‐ENaC as bona‐fide PI(3,4,5)P 3 binding sites that impinge upon channel gating rather than functioning as transducer sites coupling a binding site to the gate. PI(4,5)P 2 regulation of ENaC was not influenced by mutation of these PI(3,4,5)P 3 binding sites indicating that these different phospholipids impact channel activity at distinct binding sites. The functional PI(4,5)P 2 binding site was localized to the N‐terminus of β‐ and γ‐ENaC. Our results demonstrate that β‐ and γ‐subunits bestow phospholipid sensitivity to ENaC. Both the PI(4,5)P 2 and PI(3,4,5)P 3 binding sites in ENaC contained well conserved positive charged residues. In conclusion, β‐ and γ‐ but not α‐ENaC bind both PI(3,4,5)P 3 and PI(4,5)P 2 but at distinct sites with phospholipid binding modulating channel gating to increase open probability.