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 (Ie) that is measurable under voltage-clamp conditions3,4. Here we report the complete current–voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie 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 channels5,6 compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents7,8,9 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.
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References
Babior, B. M. NADPH oxidase: an update. Blood 93, 1464–1476 (1999)
Henderson, L. M., Chappell, J. B. & Jones, O. T. The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem. J. 246, 325–329 (1987)
Schrenzel, J. et al. Electron currents generated by the human phagocyte NADPH oxidase. Nature 392, 734–737 (1998)
DeCoursey, T. E., Cherny, V. V., Zhou, W. & Thomas, L. L. Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils. Proc. Natl Acad. Sci. USA 97, 6885–6889 (2000)
DeCoursey, T. E. & Cherny, V. V. Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils. Biophys. J. 65, 1590–1598 (1993)
Henderson, L. M., Chappell, J. B. & Jones, O. T. Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel. Biochem. J. 251, 563–567 (1988)
Mahaut-Smith, M. The effect of zinc on calcium and hydrogen ion currents in intact snail neurones. J. Exp. Biol. 145, 455–469 (1989)
Thomas, R. C. & Meech, R. W. Hydrogen ion currents and intracellular pH in depolarized voltage-clamped snail neurones. Nature 299, 826–828 (1982)
Cherny, V. V. & DeCoursey, T. E. pH-dependent inhibition of voltage-gated H+ currents in rat alveolar epithelial cells by Zn2+ and other divalent cations. J. Gen. Physiol. 114, 819–838 (1999)
Henderson, L. M., Chappell, J. B. & Jones, O. T. Superoxide generation by the electrogenic NADPH oxidase of human neutrophils is limited by the movement of a compensating charge. Biochem. J. 255, 285–290 (1988)
Price, M. O. et al. Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system. Blood 99, 2653–2661 (2002)
Morgan, D., Cherny, V. V., Price, M. O., Dinauer, M. C. & DeCoursey, T. E. Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components. J. Gen. Physiol. 119, 571–580 (2002)
Cherny, V. V., Henderson, L. M., Xu, W., Thomas, L. L. & DeCoursey, T. E. Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid. J. Physiol. (Lond.) 535, 783–794 (2001)
Nanda, A. & Grinstein, S. Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils. Proc. Natl Acad. Sci. USA 88, 10816–10820 (1991)
Reeves, E. P. et al. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416, 291–297 (2002)
Kapus, A., Szászi, K. & Ligeti, E. Phorbol 12-myristate 13-acetate activates an electrogenic H+-conducting pathway in the membrane of neutrophils. Biochem. J. 281, 697–701 (1992)
DeCoursey, T. E., Cherny, V. V., DeCoursey, A. G., Xu, W. & Thomas, L. L. Interactions between NADPH oxidase-related proton and electron currents in human eosinophils. J. Physiol. (Lond.) 535, 767–781 (2001)
Cross, A. R. & Jones, O. T. The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase. Biochem. J. 237, 111–116 (1986)
Lowenthal, A. & Levy, R. Essential requirement of cytosolic phospholipase A2 for activation of the H+ channel in phagocyte-like cells. J. Biol. Chem. 274, 21603–21608 (1999)
Koshkin, V., Lotan, O. & Pick, E. Electron transfer in the superoxide-generating NADPH oxidase complex reconstituted in vitro. Biochim. Biophys. Acta 1319, 139–146 (1997)
Burton, K. & Wilson, T. H. The free-energy changes for the reduction of diphosphopyridine nucleotide and the dehydrogenation of l-malate and l-glycerol 1-phosphate. Biochem. J. 54, 86–94 (1953)
Wood, P. M. The redox potential of the system oxygen-superoxide. FEBS Lett. 44, 22–24 (1974)
Läuger, P. Electrogenic Ion Pumps (Sinauer Associates, Sunderland, Massachusetts, 1991)
Biberstine-Kinkade, K. J. et al. Heme-ligating histidines in flavocytochrome b558: identification of specific histidines in gp91phox. J. Biol. Chem. 276, 31105–31112 (2001)
Gordienko, D. V. et al. Voltage-activated proton current in eosinophils from human blood. J. Physiol. (Lond.) 496, 299–316 (1996)
Byerly, L., Meech, R. & Moody, W. Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymnaea stagnalis. J. Physiol. (Lond.) 351, 199–216 (1984)
Tare, M. et al. Inwardly rectifying whole cell potassium current in human blood eosinophils. J. Physiol. (Lond.) 506, 303–318 (1998)
Bánfi, B. et al. A novel H+ conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease. J. Exp. Med. 190, 183–194 (1999)
Martin, M. A., Nauseef, W. M. & Clark, R. A. Depolarization blunts the oxidative burst of human neutrophils. Parallel effects of monoclonal antibodies, depolarizing buffers, and glycolytic inhibitors. J. Immunol. 140, 3928–3935 (1988)
Jankowski, A. & Grinstein, S. A noninvasive fluorimetric procedure for measurement of membrane potential. Quantification of the NADPH oxidase-induced depolarization in activated neutrophils. J. Biol. Chem. 274, 26098–26104 (1999)
Acknowledgements
We thank A. R. Cross and L. L. Thomas for discussions, and T. Iastrebova and J. Murphy for technical assistance. This work was supported in part by the Heart, Lung and Blood Institute of the National Institutes of Health (T.E.D.).
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DeCoursey, T., Morgan, D. & Cherny, V. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels. Nature 422, 531–534 (2003). https://doi.org/10.1038/nature01523
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DOI: https://doi.org/10.1038/nature01523
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