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 (IH), causing an approximately -40-mV shift in the activation threshold that leads to the appearance of inward IH. 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 [Zn2+]. IH 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 (Ie), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both IH and Ie 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 IH when a stable driving force for e- was imposed. Combined application of NADPH and DPI reduced the inward IH amplitude, even in the absence of concomitant oxidase activity. The strict correlation between Ie and I H amplitudes and the sensitivity of IH to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.
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