Proton permeation is blocked by AAA mutation and by PIP₂ addition. (A and B) The AAA SF mutation (in which the SF GYG sequence is replaced by AAA) was introduced on the I131x mutation backgrounds. All mutants show no activity in either K⁺ or Na⁺ conditions (A) and no H⁺ permeation above that for empty liposomes (Lipo) when valinomycin (Val) is added instead of CCCP (B). (C) Fluorescence assays for WT and I131S/G/M mutants in K⁺ conditions as a function of membrane [PtdIns(4,5)P₂]. All data are represented as mean ± SEM of at least n = 3 experiments.