(A1, A2) Two-minute pretreatment with mild acidification greatly suppressed I6.0 in cultured mouse cortical neurons. Shown are representative current traces at −60 mV (A1) and summary data for peak currents (A2) (n=4–6, ***p<0.001; NS, no statistical significance, vs pH 7.4). (B) Pretreatment with pH 6.8 (Pre-6.8) for 10 min failed to prevent acid-induced neuronal death (CTB assay, n=4–6, ***p<0.001, NS, no statistical significance, vs pH 7.4; n.s., no statistical significance, vs pH 6.0). Note: treatment with the pH 6.8 solution for 1 hr only did not alter neuronal viability. (C) Summary of propidium iodide (PI)-positive neurons for Asic1a+/+ and Asic1a−/− cultures treated with pH 7.4 (CTRL) or pH 6.0 solutions for 15, 30, and 60 min. Representative images are shown in Figure 3—figure supplement 1B. At least 400 neurons were counted for each condition (***p<0.001; NS, no statistical significance, vs corresponding Asic1a−/− cultures). (D) Neuronal death induced by 1 hr treatment with the pH 6.0 solution in normal (SS, standard external solution), Na+-, K+-, Ca2+-free (NMDG replacement), and Ca2+-free conditions (CTB assay, n=3, ##p<0.01 vs Na+-, K+-, Ca2+-free in pH 6.0; ***p<0.001 vs pH 7.4 under the same cation conditions). Dashed line, pH 6.0 solution-induced neuronal death under normal SS condition. (E) Representative traces of I6.0 for cortical neurons in normal (SS), Na+-, K+-, Ca2+-free and Ca2+-free conditions. (F) Representative traces of I6.0 for wild type (WT) ASIC1a and its HIF and RC mutants expressed in CHO cells. (G) Current–voltage relationship of WT-ASIC1a (filled squares) and HIF-ASIC1a (open circles) in response to the pH 6.0 solution. Note: no current was induced by pH 6.0 in CHO cells that expressed HIF-ASIC1a at a broad range of holding voltages. (H) Summary data of cell death induced by 1 hr pH 6.0 solution treatment in Asic1a−/− neurons expressing GFP vector, WT-ASIC1a, and its HIF and RC mutants, based on PI staining of GFP-labeled neurons (see Figure 3—figure supplement 6 for representative images, n=100 for each condition, ***p<0.001; NS, no statistical significance, vs pH 7.4 of the corresponding transfection; n.s., no statistical significance, vs WT-ASIC1a).