Proton fluxes across the t-system after rapid changes in cytoplasmic pH and membrane potential. In Na⁺-based internal solutions, the pH was changed from 7.7 to 7.2 to 6.9 and then changed to a K⁺-based solution with a pH of 7.5 while continuously imaging the t-system HPTS fluorescence signal. The spatially averaged profile shows a stepwise decrease in t-system pH with each drop in cytoplasmic pH in Na⁺ solution. The final solution substitution was to a pH-7.5 K⁺-based internal solution, which polarizes the t-system. (A) Fluorescence intensity and derived proton concentration ([H⁺]_t) changes in the sealed t-system. (B) Changes in total dissociable proton concentration in the sealed t-system and proton fluxes across the sealed t-system membrane.