Electrophysiology of reactive oxygen production in signaling endosomes

Abstract

Endosome trafficking and function require acidification by the vacuolar ATPase (V-ATPase). Electrogenic proton (H+) transport reduces the pH and creates a net positive charge in the endosomal lumen. Concomitant chloride (Cl-) influx has been proposed to occur via ClC Cl-=H+ exchangers. This maintains charge balance and drives Cl- accumulation, which may itself be critical to endosome function. Production of reactive oxygen species (ROS) in response to cytokines occurs within specialized endosomes that form in response to receptor occupation. ROS production requires an NADPH oxidase (Nox) and the ClC-3 Cl-=H+ exchanger. Like the V-ATPase, Nox activity is highly electrogenic, but separates charge with an opposite polarity (lumen negative). Here we review established paradigms of early endosomal ion transport focusing on the relation between the V-ATPase and ClC proteins. Electrophysiologic constraints on Nox-mediated vesicular ROS production are then considered. The potential for ClC-3 to participate in charge neutralization of both proton (V-ATPase) and electron (Nox) transport is discussed. It is proposed that uncompensated charge separation generated by Nox enzymatic activity could be used to drive secondary transport into negatively charged vesicles. Further experimentation will be necessary to establish firmly the biochemistry and functional implications of endosomal ROS production.

FIG. 1.

(A) The V-ATPase moves protons into endosomes by using energy derived from ATP hydrolysis. This results in net positive charge accumulation in the lumen of the vesicle. (B) The NADPH oxidase (Nox) transfers electrons from reduced NADPH to cytoplasmic FAD and then through two heme groups. In the lumen of the endosome, the electrons are passed to molecular oxygen to form two molecules of superoxide anion. This results in net negative charge accumulation. After regeneration of NADPH by the pentose phosphate shunt, two protons are created in the cytoplasm. The Nox protein NADPH complex is depicted without the associated membrane and cytoplasmic proteins that are required for function.

FIG. 2.

Membrane ion transporters and channels that may affect the electrophysiology of the signaling endosome. Endocytosis results in the extracellular face of these proteins being exposed to the lumen of the endosome. The intracellular ion concentrations provided for sodium (73) and Cl⁻ (14) are estimates for vascular smooth muscle cells.

FIG. 3.

(A) Charge neutralization of the V-ATPase by ClC-3. Cl⁻ enters the endosome, and H⁺ leaves with a stoichiometry of 2:1. Cl⁻/ H⁺ exchange can provide appropriate opposing current flow, but at a cost of removal of H⁺ from the compartment. The net result would be acidification and Cl⁻ accumulation. (B) Charge neutralization of the NADPH oxidase by ClC-3. H⁺ enters the endosome, and Cl⁻ leaves. Dismutation of superoxide consumes 2 H⁺ per molecule of the H₂O₂ produced. The net result of these processes would be alkalinization and Cl⁻ depletion.

FIG. 4.

Rectification of ClC-3 favors outward current in HEK 293 cells overexpressing adenoviral ClC-3. Large currents are seen at positive intracellular potentials where Cl⁻ is moving into the cell and H⁺ is moving out. After endocytosis, this becomes movement of Cl⁻ out of the signaling endosome and H⁺ movement into the vesicle. These data are reprinted from (60). *The current in ClC-3–expressing cells is significantly larger than that in control, GFP-expressing cells (p < 0.05).

FIG. 5.

Potential contributors to the electrophysiology of the signaling endosome. It remains to be determined whether the V-ATPase and Nox enzymes operate in the same or different populations of vesicles. If they are present in the same compartment, the balance between superoxide production by Nox and proton accumulation by all potential mechanisms (V-ATPase, proton channels, Na⁺/H⁺ exchange, Cl⁻/H⁺ antiport) may determine both the pH and the redox state of the compartment. Control of the microenvironment of the signaling endosome may be critical to proper processing of endocytosed proteins and subsequent signal transduction. Rapid superoxide production associated with alkalinization of the vesicle lumen could allow significant accumulation of superoxide, which may then leave the endosome via an anion conductance. This mechanism may provide a controlled pathway for cytoplasmic ROS signaling by superoxide that is independent of H₂O₂ production.

FIG. 6.

Proposed electrophysiology of the vesicle in the absence of charge neutralization. If the NADPH oxidase is activated without charge neutralization, oxidase activity, and therefore ROS production, will be low. However, a large potential difference (lumen negative) could theoretically be maintained. Under these conditions, activity of the V-ATPase should not be constrained, and a variety of secondary ion-transport mechanisms, including ion channels or co-transporters of larger charged molecules, could be facilitated. From the viewpoint of membrane proteins, this state would be the equivalent to that of a depolarized plasma membrane.