A voltage-dependent KCl HPTS base pulse assay was employed to further probe the H + /Cl -symport mechanism facilitated by this class of anionophores (Figure ). Membrane potentials were generated by varying the intravesicular and extravesicular K + concentrations and introducing valinomycin at the beginning of each experiment. Two solutions of vesicles were prepared in a solution of KCl (100 mM) or a 99:1 NaCl/KCl solution (NaCl = 99 mM, KCl = 1mM) solution, buffered to pH 7.0 with HEPES (10 mM). The LUVs were then suspended in an external solution which corresponded to the generation of a neutral membrane potential, a positive membrane potential (118 mV), or a negative potential (-118 mV). Valinomycin in a DMSO solution (5 µL, 0.1 mol%) and a NaOH base pulse (5 mM) were added to the solutions at t = -30 s before the experiment was initiated with the addition of transporter in a DMSO solution (5 µL) at t = 0 s. A single dose was used for each compound at a concentration that corresponded to the EC50 value determined in the NMDG-Cl HPTS assay. Full experimental methods and fluorescence plots can be viewed in the supplementary information.