The use of the lucigenin assay to measure chloride uniport is demonstrated in Figure using anionophore 1, which was previously shown to be an effective chloride uniporter when studied in the HPTS assay. In the absence of an anionophore, no transport of chloride is observed upon addition of a KCl pulse, even in the presence of valinomycin (black and red curves in Figure ). When 1 is pre-incorporated and valinomycin is added to facilitate potassium transport, equilibration of intra-and extravesicular chloride concentration caused by uniport of chloride by 1 is clearly observed (green curve in Figure ). In contrast, when 1 is pre-incorporated but no valinomycin is added, a smaller decrease in fluorescence intensity is observed (blue curve in Figure ). This decrease in fluorescence is larger compared to the blank experiments (without anionophore), which might be caused by a very small amount of chloride uniport (before the potential gradient stops the transport), as well as some Cl -/NO3 - antiport, due to the presence of nitrate counter anions in commercial lucigenin salt. Substituting lucigenin for SPBA as a probe for this type of experiments eliminates the Cl -/NO3 -antiport and allows for a clean observation of chloride uniport activity, as shown in Figure .