Roles of Cytoplasmic Ions in Lysosomal Acidification

Abstract

The lysosome is the terminal organelle in the endocytic pathway; its role is to degrade and recycle macromolecules. To function properly it must maintain an acidic pH. A V-type ATPase is necessary to drive protons into lysosomes, but the action of this pump generates a large voltage across the membrane, which limits further pumping. Fluxes of ions are therefore necessary to dissipate the electrochemical gradient, referred as the “counterion pathway.” It remains poorly understood which ions contribute to this pathway and facilitate net acidification of the lysosome. Our goal is to investigate the mechanism underlying the acidification of lysosomes and to evaluate the role of ClC-7, a lysosomal H+/Cl- exchanger in the acidification process. We have probed the effects of external ion concentrations on acidification in isolated lysosomes using fluorescent ion- or voltage-sensing probes to measure internal pH or changes in membrane voltage.Using isolated lysosomes we demonstrate that the presence of external anions facilitates lysosomal acidification, suggesting a role in the counterion pathway. The anion specificity is similar to the anion selectivity observed for members of the ClC family (Cl->Br->I->NO3-). In contrast, the presence of cytoplasmic like concentrations of external K+ doesn't facilitate acidification; rather, at these levels (100mM), K+ ions induce alkalinization, which is enhanced in the presence of Valinomycin. Using the DisC3(5) membrane potential dye we monitored lysosomal membrane potential while varying external [K+]; we estimate the relative K+ permeability with and without Valinomycin. Finally, we used the null point titration approach to estimate the luminal concentration of ions, obtaining values in a range similar to those found using pH changes. This work helps constrain models of lysosomal acidification and suggests a limited role for K+ in the process.