Ion selectivity of hASIC1a lower pore mutants. (A) Summaries of changes in the ion selectivity sequence, P(Na):P(K):P(Cs), of GAS belt mutants. All combinations shown have significant decreased selectivity compared with WT by one-way ANOVA post hoc Dunnett’s test (P < 0.0001), except the P(Na):P(K) ratio of GGS:SCS = 1:1 and GGS P = 0.33 and 0.026, respectively. (B) E452 and D455 mutants all display significant lower selectivity than WT (P < 0.0001). Selectivity of D455E taken from Lynagh et al. (2017). (C) H28R, W46C, L448F, and other lower pore mutants; among them, only H28R and L448F have significant lower selectivity than WT (P < 0.0001). (D) D455F rescues currents from nonfunctional mutants, but the ion selectivity of all the double mutants is low compared with WT (P < 0.0001). For measurement of reversal potential of Na⁺, K⁺, or Cs⁺ for WT and mutants, the external solution was changed from the preconditioning buffer containing 100 mM of either NaCl/KCl/CsCl, 5 mM HEPES, 5 mM Mes, and 2 mM CaCl₂, pH 7.4, to the activating buffer containing either 100 mM NaCl/KCl/CsCl, 5 mM HEPES, and 5 mM Mes, pH 6.0. Reversal potential of Na⁺, K⁺, or Cs⁺ was calculated from differences in the reversal potential of currents measured from a −80-mV to +60-mV voltage ramp by TEVC with the indicated cations in the external solutions. Data are presented as mean ± SD of two independent experiments for at least five activations from 3 to 10 Xenopus oocytes (n = 5–10) for each ion condition and for each construct tested. For hASIC1a mutants including H28R, E452Q, D455S/H/T/N, and E459F that exhibit small peak current with reduced recovery from desensitization, reversal potential of Na⁺, K⁺, or Cs⁺ was calculated only with measured activated peak current ≥3 µA/cell. Individual values and statistical analysis are shown in Data S2.