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 (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-gamma-S. A good correlation was observed between the amplitude of Ie 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 IH 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.

Pubmed ID: 14638931 RIS Download