Activation of the Human Epithelial Sodium Channel (ENaC) by the Peptidomimetic S3969 Involves a Specific Binding Pocket in the Channel’s β-Subunit

The epithelial sodium channel (ENaC) mediates Na+ absorption in several epithelia. Its impaired function leads to severe disorders, like pseudohypoaldosteronism type 1 and respiratory distress. Interestingly, a small molecule ENaC activator, the peptidomimetic S3969, stimulates human but not mouse αβγ-ENaC (Lu et al. 2008, J Biol Chem). Our aim was to identify and characterize the S3969 binding site in human ENaC. ENaC was heterologously expressed in Xenopus laevis oocytes, amiloride-sensitive whole-cell currents (ΔIami) were measured using the two-electrode voltage clamp technique. A putative S3969 binding site was predicted by using molecular docking and molecular dynamics (MD) simulations based on a recently published ENaC structure (Noreng et al. 2020, Elife). In addition, the effect of S3969 on endogenously expressed ENaC was studied in human airway epithelial cells (H441) using transepithelial measurements in Ussing chambers. We confirmed that in oocytes S3969 activated human αβγ-ENaC by ~2-fold with an EC50 of ~0.3 μM (n=25). As reported, murine αβγ-ENaC was insensitive to S3969 in concentrations up to 10 μM (n=19). Using mouse-human chimeric ENaC, we found that a portion of the extracellular loop of the β-subunit (residues 256-404), which includes parts of the thumb, palm and β-ball domains, is critical for channel stimulation by S3969. Computer simulations predicted a putative S3969 binding pocket in this area, with βR388, βF391, and βY406 as key residues coordinating S3969. Mutating these residues strongly reduced (βR388A) or nearly abolished (βY406A; βF391G) the stimulatory effect of S3969 on ΔIami (n=12-18), without altering the effect of an alternative ENaC activator chymotrypsin (n=5). MD simulations also suggested that the binding of S3969 to ENaC increased the distance between the β-thumb and the γ-palm domain. Consistent with this, the stimulatory effect of S3969 was abolished when the β-thumb domain was covalently attached to the γ-palm domain following the introduction of two cysteine residues (βR437C, γS298C) to form a disulfide bridge (n=15). Importantly, reducing the disulfide bond with DTT partially rescued the effect of S3969 on ΔIami (relative stimulation 31±1%, n=15, p<0.001). We also showed that S3969 stimulated endogenously expressed human ENaC in H441 cells. In conclusion, we demonstrated that S3969 activates ENaC by interacting with a specific binding pocket in β-ENaC which probably increases the β-γ-intersubunit distance. These results may help to identify novel ENaC modulators with potential physiological or therapeutic implications. Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project number 509149993, TRR 374 (subproject A4 to A.I. and C.K.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.