A hydrogen bond between W46 and E452 stabilizes the predicted open conformation. (A) Side view of the predicted hASIC1a in the resting (left panel) and open (right panel) conformations, highlighting interaction networks formed by residues E452 (TM2b), W46 (TM1), T26 (HG loop), and H32 (Re-2). Only two subunits are shown. TMs are in cyan/pale cyan, while corresponding reentrant loops are in violet/light pink. (B) Summaries of normalized current, τ_d, and recovery ratio for E452. (C and D) W46 (C) and H32 (D) substitutions. (E) Representative traces of E452R, W46C, H32R, H32L, and H32Y. Data are presented as mean ± SD of three independent experiments for at least five Xenopus oocytes (n = 5–10) for each construct tested. All oocytes were incubated at least 30 s (30–40 s) in preconditioning buffer (pH 7.4) for channel recovery before the next activation. Recovery ratio (expressed in percent) was calculated as I_{acti(n + 1)}/I_acti(n). Individual measurements and statistical analysis are shown in Data S2.