Epithelial Na+ channel (ENaC) gating is allosterically modulated by both its extracellular and intracellular domains. Among several regions that have been implicated in regulating ENaC gating are the extracellular palm domains where three subunits interface. We previously identified a palm domain hydrophobic residue (γL511) as an important determinant of ENaC gating. Interestingly, its homologous residue in chicken ASIC1a (L414), located at the β11 and β12 linker, reportedly adapts flipped conformations in the rest (or open) and desensitized states. In this study, we investigated the roles of γL511 homologous resides in the α and β subunits (αL531 and βF502) in regulating ENaC gating, by mutating these residues to a hydrophilic glutamine and examining channel properties in Xenopus oocytes. ENaCs both transport Na+ and are inhibited by extracellular Na+, a response referred to as Na+ self‐inhibition that reflects a reduction in channel open probability and is often used as a surrogate for ENaC gating. Similar to γL511Q, both αL531Q and βF502Q significantly reduced the magnitude and speed of the Na+ self‐inhibition response (p<0.01 vs WT, n=6). Oocytes expressing αL531Qbg ENaCs also showed significantly greater amiloride‐sensitive currents (relative‐to‐WT currents: 1.9 ± 0.2, Mean ± SE, p<0.001, n=32). However, βF502Q expressing oocytes showed the channel currents that were similar to wild type (relative‐to‐WT currents: 0.9 ± 0.1, p>0.05, n=22). The changes caused by both αL531Q and βF502Q on the channel activity and Na+ self‐inhibition response appear to be less dramatic than the previously reported γL511Q. Our results suggest that the highly conserved palm domain hydrophobic residues in three subunits have similar roles in regulating ENaC gating.Support or Funding InformationDK079307 DK051391This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.