Cytosolic cations support protonophore-induced lysosome alkalinization. (A) Schematic representation of the experimental protocol used to dialyze and alkalinize the lysosomes. After baseline pH measurements (step I), the cytosol was dialyzed by selectively permeabilizing the plasma membrane through activation of P2X₇ receptors in the presence of suitable buffers (step II). Removal of the P2X₇ agonists and the addition of divalent cations resealed the plasma membrane (step III). The lysosomes were then alkalinized by FCCP treatment (step IV). (B) Proton efflux from lysosomes requires either the parallel efflux of an anion or the influx of a cation. When protonophore (green star) is added, counter-ions must move across the lysosome membrane to prevent membrane potential buildup. Before addition of the protonophore, the cytosol was dialyzed in the presence of either K⁺, Na⁺ (left) or NMDG⁺ (right), a relatively impermeant cation. If a K⁺ and/or Na⁺ conductance exists, NMDG⁺ will impair lysosome alkalinization, as it cannot be effectively transported into the lysosome lumen to dissipate the voltage. (C) Lysosome alkalinization in cells dialyzed with K⁺-rich (diamond), Na⁺-rich (squares), or NMDG⁺-rich solution (triangles). The time course of the change in pH after FCCP addition (arrow) is illustrated for representative experiments (means ± SD). (D) The rate of pH dissipation over the first minute of FCCP-induced alkalinization for the three different cations.