Thiamine transport by PnuT_Sw reconstituted in proteoliposomes. (a) Transport measured at different voltages across the liposomal membrane: −110 mV (black circles), −55 mV (open circles), −40 mV (black triangles), −22 mV (open triangles), and 0 mV (black squares). The gray dashed line shows the thiamine association to protein-free liposomes (membrane voltage −110 mV). In the latter case, the scale of the y-axis is pmol of thiamine per 250 µg lipids. (b) Transport measured at different external pH values: pH 5.5 (squares), pH 6.5 (circles), pH 7.5 (triangles), and pH 8.5 (inverted triangles). The internal pH was 7.5 in all cases. (c) Transport measured in the presence (open circles) of absence (black circles) of external Na⁺. In the latter case, sodium chloride was replaced by choline chloride. (d) Demonstration of bidirectionality of thiamine transport. The luminal and external pH were identical (pH 7.5). Closed circles: The membrane voltage was clamped at −110 mV using valinomycin at the onset of the experiment, and FCCP was added after six minutes. Open circles: Valinomycin was added only after 6 min. Inverted triangles: No valinomycin was present, and FCCP was added after 6 min. All error bars indicate the SD from three experiments.