Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase

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, I_e, that are associated with inward proton currents, I_H. To study the inactivation of the phox and of its associated proton channel, we determined which soluble factors can stabilize I_e (induced by the addition of NADPH) and I_H (initiated by small depolarizing voltage steps) in inside-out patches from PMA-activated human eosinophils. I_e 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. I_H also decayed rapidly and was stabilized by the ATP/GTP[S] mixture, but maximal stabilization of I_H 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 I_e and its stabilizing effect on I_H could not be mimicked by other phosphoinositides. Reducing the ATP concentration below millimolar levels decreased I_H 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 p47^phox to the plasma membrane.