The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

Abstract

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O₂^-, precursors to bactericidal reactive oxygen species¹). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O₂ to O₂^-. NADPH oxidase is electrogenic², generating electron current (I_e) that is measurable under voltage-clamp conditions^3,4. Here we report the complete current–voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I_e is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H⁺ efflux² mediated by voltage-gated proton channels^5,6 compensates I_e, because Zn²⁺ and Cd²⁺ inhibit both H⁺ currents^7,8,9 and O₂^- production¹⁰. Here we show that COS-7 cells transfected with four NADPH oxidase components¹¹, but lacking H⁺ channels¹², produce O₂^- in the presence of Zn²⁺ concentrations that inhibit O₂^- production in neutrophils and eosinophils. Zn²⁺ does not inhibit NADPH oxidase directly, but through effects on H⁺ channels. H⁺ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.