Substitutions at the GAS or L448 alter channel kinetics by destabilizing the three states. (A) Summaries of normalized current, τ_d, and recovery ratio for GAS and L448 substitutions. 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 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 values are shown in Data S2. (B) Free energy change (ΔΔG) calculations of the above substitutions in the closed, open, and desensitized states compared with the WT hASIC1a in the corresponding conformations. A ΔΔG value > 1 is considered to be destabilizing, while a ΔΔG value less than −1 is considered to be stabilizing. All values are presented as mean ± SD. 10 runs of free energy calculations were conducted in replicates for each mutant and the WT hASIC1; 10 free energy values of the WT and all mutants were sorted from smallest to largest before free energy values of the WT were subtracted from those of each mutant (10 ΔΔG values obtained for each mutant). Individual values are provided in Data S2.