To further elucidate the mechanism of chloride transport by 2, Cl -/SO4 2-exchange was attempted. Due to its double charge and extreme hydrophilicity, SO4 2-is considered unable to pass through lipid bilayers without the help of specialized transporters, and therefore unlikely to exchange with Cl -via an antiport mechanism. Somewhat surprisingly, we observed a small (approx. 10%), albeit significant drop in fluorescence intensity within the first ca. 100 s, followed by a plateau (Fig. ). We hypothesized that this is due to H + /Cl -co-transport, which generates an H + gradient across the membrane and, in this way, prevents further chloride influx. Alternative mechanisms, such as Cl -/SO4 2-exchange and Na + /Cl -co-transport, would lead to full equilibration between the interior and exterior of the LUVs. The latter two mechanisms were also ruled out based on HPTS assays performed in sodium gluconate and in N-methyl-D-glucamine sulfate solutions (see SI). The H + /Cl -co-transport in sodium sulfate solution was further confirmed using vesicles loaded with a pH-sensitive dye, HPTS, instead of lucigenin. Upon injection of NaCl, a decrease of the intravesicular pH was observed, indicative of the transport of H + along Cl -into the vesicles (Fig. ).