The phagocytic NADPH oxidase (phox) moves electrons across cell membranes to kill microbes. The activity of this lethal enzyme is tightly regulated, but the mechanisms that control phox inactivation are poorly understood for lack of appropriate assays. The phox generates measurable electron currents, Ie, that are associated with inward proton currents, IH. To study the inactivation of the phox and of its associated proton channel, we determined which soluble factors can stabilize Ie (induced by the addition of NADPH) and IH (initiated by small depolarizing voltage steps) in inside-out patches from PMA-activated human eosinophils. Ie decayed rapidly in the absence of nucleotides (τ≈6 min) and was maximally stabilized by the combined addition of 5 mM ATP and 50 μM of the non-hydrolysable GTP analogue GTP[S] (guanosine 5′-[γ-thio]triphosphate) (τ≈57 min), but not by either ATP or GTP[S] alone. IH also decayed rapidly and was stabilized by the ATP/GTP[S] mixture, but maximal stabilization of IH required further addition of 25 μM PI(3,4)P2 (phosphoinositide 3,4-bisphosphate) to the cytosolic side of the patch. PI(3,4)P2 had no effect on Ie and its stabilizing effect on IH could not be mimicked by other phosphoinositides. Reducing the ATP concentration below millimolar levels decreased IH stability, an effect that was not prevented by phosphatase inhibitors but by the non-hydrolysable ATP analogue ATP[S] (adenosine 5′-[γ-thio]triphosphate). Our data indicate that the assembled phox complex is very stable in eosinophil membranes if both ATP and GTP[S] are present, but inactivates within minutes if one of the nucleotides is removed. Stabilization of the phox-associated proton channel in a highly voltage-sensitive conformation does not appear to involve phosphorylation but ATP binding, and requires not only ATP and GTP[S] but also PI(3,4)P2, a protein known to anchor the cytosolic phox subunit p47phox to the plasma membrane.
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Research Article|
November 28 2006
Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase
Gábor L. Petheő;
Gábor L. Petheő
1
*Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland
†Department of Physiology and Laboratory of Cellular and Molecular Physiology, Faculty of Medicine, Semmelweis University, P.O. Box 259, H-1444 Budapest, Hungary
1To whom correspondence should be addressed (email petheo@puskin.sote.hu).
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Nathalie C. Girardin;
Nathalie C. Girardin
*Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland
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Nicolas Goossens;
Nicolas Goossens
*Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland
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Gergely Z. Molnár;
Gergely Z. Molnár
*Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland
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Nicolas Demaurex
Nicolas Demaurex
*Department of Cell Physiology and Metabolism, University of Geneva Medical School, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland
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Publisher: Portland Press Ltd
Received:
April 19 2006
Revision Received:
July 07 2006
Accepted:
July 14 2006
Accepted Manuscript online:
July 14 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 400 (3): 431–438.
Article history
Received:
April 19 2006
Revision Received:
July 07 2006
Accepted:
July 14 2006
Accepted Manuscript online:
July 14 2006
Citation
Gábor L. Petheő, Nathalie C. Girardin, Nicolas Goossens, Gergely Z. Molnár, Nicolas Demaurex; Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase. Biochem J 15 December 2006; 400 (3): 431–438. doi: https://doi.org/10.1042/BJ20060578
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