Determinants of [Cl⁻] in recycling and late endosomes and Golgi complex measured using fluorescent ligands

Chloride concentration ([Cl⁻]) was measured in defined organellar compartments using fluorescently labeled transferrin, α₂-macroglobulin, and cholera toxin B-subunit conjugated with Cl⁻-sensitive and -insensitive dyes. In pulse-chase experiments, [Cl⁻] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ∼10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl⁻] just after internalization (compared with 137 mM solution [Cl⁻]) was prevented by reducing the interior-negative Donnan potential. [Cl⁻] in α₂-macroglobulin–labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1–45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl⁻ accumulation was prevented by bafilomycin but restored by valinomycin. A Cl⁻ channel inhibitor slowed endosomal acidification and Cl⁻ accumulation by ∼2.5-fold. [Cl⁻] was 49 mM and pH was 6.42 in cholera toxin B subunit–labeled Golgi complex in Vero cells; Golgi compartment Cl⁻ accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl⁻ is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl⁻] early after internalization. We propose that reduced [Cl⁻] and volume in early endosomes permits endosomal acidification and [Cl⁻] accumulation without lysis.