Interactions between Electron and Proton Currents in Excised Patches from Human Eosinophils

The NADPH–oxidase is a plasma membrane enzyme complex that enables phagocytes to generate superoxide in order to kill invading pathogens, a critical step in the host defense against infections. The oxidase transfers electrons from cytosolic NADPH to extracellular oxygen, a process that requires concomitant H(+) extrusion through depolarization-activated H(+) channels. Whether H(+) fluxes are mediated by the oxidase itself is controversial, but there is a general agreement that the oxidase and H(+) channel are intimately connected. Oxidase activation evokes profound changes in whole-cell H(+) current (I (H)), causing an approximately −40-mV shift in the activation threshold that leads to the appearance of inward I (H). To further explore the relationship between the oxidase and proton channel, we performed voltage-clamp experiments on inside-out patches from both resting and phorbol-12-myristate-13-acetate (PMA)-activated human eosinophils. Proton currents from resting cells displayed slow voltage-dependent activation, long-term stability, and were blocked by micromolar internal [Zn(2+)]. I (H) from PMA-treated cells activated faster and at lower voltages, enabling sustained H(+) influx, but ran down within minutes, regaining the current properties of nonactivated cells. Bath application of NADPH to patches excised from PMA-treated cells evoked electron currents (I (e)), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both I (H) and I (e) was delayed, and sometimes prevented, by cytosolic ATP and GTP-γ-S. A good correlation was observed between the amplitude of I (e) and both inward and outward I (H) when a stable driving force for e(−) was imposed. Combined application of NADPH and DPI reduced the inward I (H) amplitude, even in the absence of concomitant oxidase activity. The strict correlation between I (e) and I (H) amplitudes and the sensitivity of I (H) to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.