Mutations at position I131 alter KirBac1.1 ion selectivity and generate proton translocation. (A) Cartoon diagram of ACMA fluorescence-based assays with KirBac1.1 channels embedded in liposomes. (1) ACMA/150 mM NMDG solution. Maximal ACMA fluorescence is present due to no liposome addition. (2) Liposomes that are generated with high (150 mM) Na⁺ or K⁺ on both sides are diluted in the ACMA/150 mM NMDG solution. Solution is switched to low (nominally zero) Na⁺ or K⁺. ACMA fluorescence is maintained, unless counterion flow allows Na⁺ or K⁺ efflux. (3) Addition of CCCP allows counterion entry of protons to quench ACMA fluorescence, in proportion to Na⁺ or K⁺ efflux. (4) Monensin or Valinomycin addition allows maximal Na⁺ or K⁺ efflux, respectively. (B) K⁺-driven (left) and Na⁺-driven (right) fluxes. WT KirBac1.1 K⁺ and Na⁺ fluxes (black line) are minimal unless CCCP is added. I131S/G/M (red, blue, and green lines, respectively) mutants translocate protons in absence of CCCP and increase channel-dependent Na⁺ fluxes. (C) Relative flux calculated from B at the end of the low-Na/K phase (2; light color) and at the end of CCCP phase (3; dark), normalized to maximum ACMA fluorescence in high [K⁺/Na⁺] (1) and minimum fluorescence in Val/Mon (4) after subtraction of empty liposome values. (D) Relative flux calculated as in C, for additional I131 mutants, as indicated. Amino acids are grouped as hydrophobic (orange, Φ), hydrophilic (blue, ζ), negatively charged (green, –), positively charged (purple, +), and aromatic (light blue, Ω). All data are represented as mean ± SEM of at least n = 3 experiments.