Interactions between electron and proton currents in excised patches from human eosinophils

Area of Science:

• Cellular Biology
• Immunology
• Biochemistry

Background:

• The NADPH-oxidase is crucial for host defense, generating superoxide to kill pathogens.
• This process involves electron transfer and requires proton (H+) extrusion through associated channels.
• The precise relationship between the NADPH-oxidase and H+ channels remains debated.

Purpose of the Study:

• To investigate the functional relationship between the NADPH-oxidase and H+ currents in human eosinophils.
• To determine if H+ fluxes are directly mediated by the oxidase complex or linked to it.

Main Methods:

• Utilized voltage-clamp experiments on inside-out patches from resting and PMA-activated human eosinophils.
• Measured proton currents (IH) and electron currents (Ie) evoked by NADPH.
• Assessed the effects of inhibitors (Zn2+, DPI) and signaling molecules (ATP, GTP-gamma-S) on currents.

Main Results:

• Oxidase activation by PMA altered IH characteristics, causing faster activation and inward currents.
• Both IH and electron currents (Ie) from activated cells exhibited rapid rundown, which was delayed by ATP/GTP-gamma-S.
• A strong correlation was found between Ie and IH amplitudes, and IH was sensitive to oxidase inhibitors.

Conclusions:

• The findings strongly suggest that the proton channel is either an integral component of the NADPH-oxidase complex or intimately linked to it.
• This close association is critical for efficient superoxide generation and host defense.
• The regulation of these currents by nucleotides points to a role for intracellular signaling in oxidase function.