Fig. 2. Functional analysis of fluorophore-labeled KirBac1.1 cysteine-substituted mutants.

Fluorophore-labeled KirBac1.1 mutants were reconstituted into liposomes (POPE:POPG=3:1) with or without 1.25% PIP₂ at protein/lipid ratio of 1:100 (w/w). The intraliposome buffer was 10 mM HEPES, 450 mM KCl and 4 mM NMDG, pH7.5, and the extraliposome buffer was 10 mM HEPES, 50 mM KCl, 400 mM sorbitol and 4 mM NMDG, pH7.5. ⁸⁶Rb⁺ uptake was measured at 15 min and normalized against the maximal ⁸⁶Rb⁺ uptake in the presence of valinomycin (Rb uptake). ⁸⁶Rb⁺ uptake of fluorophore-labeled mutants is shown as ⁸⁶Rb⁺ flux relative to wild type (mean±S.E, n=3 in each case). Background level of ⁸⁶Rb⁺ uptake (in liposomes with no protein) is marked by a red dashed line.