A π–π interaction between F21 and Y456 stabilizes the resting conformation. (A) Top view of the predicted hASIC1a in the closed and open conformations shows a π–π interaction between the conserved residues F21 (TM1) and Y456 (TM2b) in the resting state but not in the open conformation. TM2bs from the three ASIC1 subunits are presented in deep teal/cyan/pale cyan, while the three corresponding reentrant loops are displayed in light magenta/violet/light pink. (B) Summaries of normalized current, τ_d, and recovery ratio of F21 substitutions. (C) From Y456 substitutions, only Y456W produced peak currents of a magnitude similar to WT (Dunnett’s P = 0.9), whereas all other mutants had P < 0.0001. (D) Representative current traces of WT hASIC1a, F21W, Y456H, and Y456C. Data are presented as mean ± SD of three independent experiments for at least five Xenopus oocytes (n = 5–10) for each construct tested. Oocytes were incubated for 30 s in preconditioning buffer (pH 7.4) before a second activation to measure the recovery ratio (expressed in percent), calculated as I_{acti(n + 1)}/I_acti(n). Individual measurements and statistical analysis are shown in Data S2.