Figure 2. PAC is required for an acid-sensitive Cl⁻ conductance in endosomes.

(A) Diagrams illustrating the technique and nomenclature used in whole-endosome recording. Top: endosomes are enlarged by transiently transfecting HEK293T cells with mCherry-tagged Rab5-Q79L, which is colocalized with endogenous PAC. Scale bar, 5 μm. Bottom: enlarged endosomes are individually released from Rab5-Q79L-transfected cells ruptured by a glass pipette and are subject to whole-endosome recording (Chen et al., 2017). Δψ is the electrical potential across the endosomal membrane, with lumen used as the reference (Bertl et al., 1992; Cang et al., 2015). Outward currents are plotted as positive values, representing the movement of negative charges (Cl⁻) out of the endosomal lumen into the topological space equivalent to cytosol (bath).

(B–D) Acid-sensitive currents monitored by voltage-ramp (left) and voltage-step (right) protocols in WT endosomes: (B) (pH 5.5) 150 mM luminal and cytosolic Cl⁻; (C) (pH 5.5) 1 mM luminal and 150 mM cytosolic Cl⁻; and (D) (pH 7.2) 150 mM luminal and cytosolic Cl⁻.

(E) Acid-sensitive currents monitored by voltage-ramp (left) and voltage-step (right) protocols in PAC KO endosomes (pH 5.5; 150 mM luminal and cytosolic Cl⁻).

(F) Current amplitudes of (B)–(E) at +100 mV. Error bars represent mean ± SEM.