Cation selectivity of Ae4. (A) Changes in pH_i seen in response to a reduction in the external [Cl⁻] was tested in Ae4-transfected cells when Na⁺ (black circles; n = 10; change from solution A to B [Table 1]), K⁺ (green circles; n = 8; change from solution J to K [Table 1]), or NMDG (blue circles; n = 6; change from solution C to D [Table 1]) was the main cation in the external solutions. (B) Summary of experiments shown in A for Na⁺ and K⁺, as well as those with external solutions containing Rb⁺ (n = 6), Li⁺ (n = 6), and Cs⁺ (n = 6; 100 mM each; see solutions Rb⁺ 100, Li⁺ 100, and Cs⁺ 100 in Table 3). For Cs⁺, the white bar is significantly greater, “a” compared with “b” (P < 0.05, one-way ANOVA followed by Bonferroni’s post hoc test). (C) Cartoon depicting the experimental design used to evaluate K⁺ transport by Ae4. (D) Nontransfected (green circles; n = 8) and Ae4-transfected cells (black circles; n = 6) loaded with the K⁺ indicator PBFI-AM were initially depleted of Cl⁻ by incubating the cells with a low Cl⁻ (solution K [Table 1]) and then Cl⁻ uptake activated by switching to an external solution containing high Cl⁻ (solution C [Table 1]). The experiment was conducted under Na⁺-free conditions to prevent Na⁺ contamination of the PBFI signal. (A, B, and D) Results are presented as the mean ± SEM.