ENaC is a heterotrimeric ion channel typically formed by α-, β-, and γ-subunits [1, 2]. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α- and γ-ENaC [1-3]. In good agreement with this, applying synthetic inhibitory peptides corresponding to key inhibitory sequences within the α- or γ-inhibitory tracts was shown to reduce ENaC activity [4, 5]. Recently published structural data of ENaC (PDB-ID: 6WTH; [2]) provided information regarding the putative binding pockets of the inhibitory peptides. However, the functional importance of individual residues belonging to these binding pockets is still incompletely understood. We performed atomistic molecular dynamic (MD) simulations to predict critical interactions between the inhibitory peptides and their binding sites in α- and γ-ENaC. Computer predictions were verified by combining site-directed mutagenesis with two-electrode voltage clamp current measurements of human wild-type or mutant αβγ-ENaC heterologously expressed in Xenopus laevis oocytes. ENaC function was assessed by measuring amiloride-sensitive currents (ΔI_ami). Values are presented as mean ± SEM. One-way ANOVA with Bonferroni posthoc test was used for statistical analysis. MD simulations demonstrated that α- and γ-inhibitory peptides form stable interactions with a central hydrophobic patch in their respective binding site. Interestingly, these hydrophobic patches are formed by four amino acid residues, which are conserved in α- (α_F226, α_W251, α_H255, α_Y447) and γ-ENaC (γ_F204, γ_W229, γ_H233, γ_Y425). To assess the functional importance of these residues, we individually replaced them by an alanine and compared the effects of synthetic α or γ inhibitory peptides on channels with corresponding mutations in α or γ ENaC with those on wild-type ENaC. Except for α_F226Aβγ- and αβγ_Y425A-ENaC, which for unknown reasons could not be functionally expressed, all other ENaC mutants produced measurable ΔI_ami in oocytes. Compared to its inhibitory effect on wild-type ENaC (−76±2 %, n=41) the inhibitory effect of the synthetic α-peptide (30 µM) was strongly reduced in α_W251Aβγ-ENaC (−28±4 %, n=14, p<0.001) and to lesser extents in α_H255Aβγ-ENaC (−61±4 %, n=12, p<0.001) and α_Y447Aβγ-ENaC (−47±4 %, n=14, p<0.001). To investigate the effect of the γ-inhibitory peptide on ENaC, oocytes were pre-treated with chymotrypsin to remove the endogenous γ-inhibitory tract, which otherwise would have occupied its binding site in γ-ENaC. Compared to its inhibitory effect on wild-type ENaC (−58±2 %, n=44), the inhibitory effects of the synthetic γ-peptide (10 µM) on αβγ_F204A-ENaC (−15±1 %, n=13, p<0.001), αβγ_W229A– (−17±6 %, n=12, p<0.001) and αβγ_H233A-ENaC (−24±1 %, n=17, p<0.001) were significantly reduced. Consistent with these findings, we found that hydrophobic amino acid residues belonging to the endogenous γ-ENaC inhibitory tract are critical for its inhibitory effect on the channel. The leucine residue γ_L160, which is expected to interact with γ_W229, appeared to be particularly important. Its substitution by an alanine increased baseline currents by ~2-fold, mimicking the stimulatory effect of chymotrypsin on the channel. These findings highlight the functional importance of conserved hydrophobic interactions of synthetic inhibitory peptides or endogenous inhibitory tracts with their corresponding binding pockets in α- and γ-ENaC. This adds to our molecular understanding of proteolytic ENaC activation.