Additional experiments were undertaken to identify the proton transport mechanism, and the influence of fatty acids in the lipid bilayer on the rate of transport (Figure ). POPC vesicles were prepared with a solution of NaCl (100 mM) and HPTS (1mM), buffered to pH 7.0 with HEPES (5 mM). The vesicles were suspended in an identical external solution which was prepared without the presence of HPTS. A pH gradient was induced with a NaOH base pulse (5 mM) and transporter was added to the experiment as a DMSO solution (5 µL to a total volume of 2.5 mL) at t = 0 s. A single dose was used for each compound corresponding to the concentration used in the voltage dependent HPTS assays. The experiments were repeated in the presence of carbonyl cyanide-mchlorophenylhydrazone (CCCP), with the protonophore being added at t = -30 s.