Ion permeability determined by ACMA-based cation flux assay. (a) KtrB-containing liposomes at an LPR of 100:1 loaded with NaCl (orange), KCl (purple), RbCl (red), or CsCl (blue) were diluted 1:20 into LiCl-based buffer. Empty liposomes filled with the respective cations are shown as a control (light colors). The addition of H⁺ ionophore CCCP is indicated by the number sign (#). Consequently, the efflux of ions was accompanied by an intravesicular pH decrease, which quenched the fluorescent dye ACMA. Finally, for the normalization (norm.) of all data, sodium ionophore IV (NaI IV) for NaCl-containing liposomes or Val for all other intravesicular cations was added, indicated by the asterisk (*). This induced protein-independent cation efflux leading to maximal ACMA quenching. (b) Fluorescence (FL) change (%) of proteoliposomes and empty liposomes at 1,800 s after CCCP addition are shown in a bar graph (n ≥ 3, ±SD). (c) KtrB_G316S-containing liposomes at an LPR of 100:1 loaded with NaCl (orange), KCl (purple), RbCl (red), or CsCl (blue) were diluted 1:20 into LiCl-based buffer. Experiments were performed as described above. (d) Decay rates (⁠$k=1/t1$⁠) for one-phase exponential decays (⁠$y=y0+A−xt$⁠) of Na⁺, K⁺, and Rb⁺ fluxes mediated by wild-type (wt) KtrB and KtrB_G316S, respectively, are shown in a bar graph (n ≥ 3, ±SD).