Proton cotransport is metal specific. (A) Schematic for detecting H⁺ influx into proteoliposomes at various ΔΨ values, pH 7.0 on both sides, in the absence or presence of 750 µM metal substrate. The setup was identical to that used in Fig. 2 (A and B), except the pH-sensitive dye BCECF replaced the metal-sensitive dye Fura-2. (B) Representative time traces (n = 8) of H⁺ uptake into proteoliposomes. Negative ΔΨ drove DraNramp H⁺ import. Mn²⁺ and Co²⁺ stimulated H⁺ entry, while Cd²⁺ did not and instead reduced H⁺ influx at ΔΨ = −120 mV. (C) Initial metal (black) and H⁺ (color) uptake rates show that Mn²⁺, Fe²⁺, and Co²⁺ transport stimulated H⁺ influx above its basal no-metal rate, while Cd²⁺ and Zn²⁺ transport did not. Stoichiometry ratios (numbers above bars) were calculated for Cd²⁺ and Mn²⁺ and were ∼1:1 for Mn²⁺:H⁺ transport in the presence of metal. Data are averages ± SEM (n ≥ 4). See also Fig. S3.