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

Abstract

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 (tau approximately 6 min) and was maximally stabilized by the combined addition of 5 mM ATP and 50 microM of the non-hydrolysable GTP analogue GTP[S] (guanosine 5'-[gamma-thio]triphosphate) (tau approximately 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 muM 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'-[gamma-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.

Voltage-gated proton channels

“…The enhanced gating mode was preserved only by a combination of 5 mM ATP, 50 µM ATPγ-s, and 25 mM phosphoinositide 3,4-bisphosphate [152]. Under similar conditions, we do not consistently observe rundown; however, it may be that our excised patches include submembrane components that preserve function.…”

“…The “activated” state of proton currents, which is discussed below, was reported to exhibit rapid spontaneous rundown ( i.e ., reversion to the “resting” state) in excised inside-out membrane patches from eosinophils [57, 152]. The enhanced gating mode was preserved only by a combination of 5 mM ATP, 50 µM ATPγ-s, and 25 mM phosphoinositide 3,4-bisphosphate [152].…”

VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

“…The current-voltage relationship of control neutrophils was outwardly rectifying with a threshold of activation around 30 mV, whereas the I-V curve of VSOP/Hv1 / neutrophils was completely flat. We and others previously suggested that the phagocyte NADPH oxidase contains a built-in proton channel (Henderson et al, 1987) that opens only when the oxidase is active (Bánfi et al, 1999;Petheo et al, 2006). Whether the oxidase is a proton channel or modulates another channel has been much debated (Maturana et al, 2002;Morgan et al, 2002;Touret and Grinstein, 2002).…”

Voltage‐gated proton channels: what's next?

“…The stability of the enhanced gating mode of proton channels in inside‐out patches appears to be supported by a combination of ATP, GTPγS and PI(3,4)P 2 (phosphoinositide 3,4‐bisphosphate). The mechanism was hypothesized to include interaction with the p47 phox component of NADPH oxidase (Petheö et al 2006), adding to the intriguing question of the nature of the relationship between proton channels and NADPH oxidase. The details of the interactions of these regulatory molecules with proton channels need further clarification.…”