Interactions between NADPH oxidase‐related proton and electron currents in human eosinophils

Abstract

Proton and electron currents in human eosinophils were studied using the permeabilized‐patch voltage‐clamp technique, with an applied NH4+ gradient to control pHi. Voltage‐gated proton channels in unstimulated human eosinophils studied with the permeabilized‐patch approach had properties similar to those reported in whole‐cell studies. Superoxide anion (O2−) release assessed by cytochrome c reduction was compared in human eosinophils and neutrophils stimulated by phorbol myristate acetate (PMA). PMA‐stimulated O2 release was more transient and the maximum rate was three times greater in eosinophils. In PMA‐activated eosinophils, the H+ current amplitude (IH) at +60 mV increased 4.7‐fold, activation was 4.0 times faster, deactivation (tail current decay) was 5.4 times slower, the H+ conductance‐voltage (gH‐V) relationship was shifted ‐43 mV, and diphenylene iodinium (DPI)‐inhibitable inward current reflecting electron flow through NADPH oxidase was activated. The data reveal that PMA activates the H+ efflux during the respiratory burst by modulating the properties of H+ channels, not simply as a result of NADPH oxidase activity. The electrophysiological response of eosinophils to PMA resembled that reported in human neutrophils, but PMA activated larger proton and electron currents in eosinophils and the response was more transient. ZnCl2 slowed the activation of H+ currents and shifted the gH‐V relationship to more positive voltages. These effects occurred at similar ZnCl2 concentrations in eosinophils before and after PMA stimulation. These data are compatible with the existence of a single type of H+ channel in eosinophils that is modulated during the respiratory burst.

A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils

“…Human basophils voltage-clamped in the perforated-patch configuration exhibit voltage-gated proton currents (Fig. 1A) that resemble those in human eosinophils (7), both in properties and in amplitude. In basophils at symmetrical pH 7.0 (50 mM NH 4 ϩ on both sides of the membrane clamps pH i near pH o ), the proton conductance, g H , activates during depolarizing voltage pulses above ϩ20 mV, and activates more rapidly at more positive voltages.…”

“…In phagocytes, they are thought to enable sustained NADPH oxidase activity by compensating for the electrogenic activity of the oxidase (3)(4)(5). Stimuli that activate NADPH oxidase in human eosinophils and neutrophils and in murine osteoclasts greatly enhance the opening of proton channels in these cells studied in perforated-patch configuration (6)(7)(8)(9)(10), largely via PKC phosphorylation (11). In contrast, phorbol myristate acetate (PMA) has no clear effect on proton currents in alveolar epithelial cells that lack NADPH oxidase (6).…”

Regulation of eosinophil membrane depolarization during NADPH oxidase activation

“…Although originally thought to be part of the NADPH oxidase complex, the proton channel now appears to be a distinct protein that is, nevertheless, concomitantly regulated by PKC (DeCoursey et al, 2001a). Stimuli such as arachidonic acid and PMA, which activate the proton channels in neutrophils and eosinophils, also stimulate electron transport through the oxidase (DeCoursey et al, 2000;DeCoursey et al, 2001b;Cherny et al, 2001). Although PMAstimulated NADPH oxidase activity can be blocked with the PKCδ selective inhibitor, rottlerin, the proton conductance is insensitive to PKCδ inhibition (Bankers-Fulbright, 2001).…”

“…This is consistent with recent results obtained in neutrophils (Jankowski and Grinstein, 1999). Although several groups have described the presence of proton channels in eosinophils (Gordienko et al, 1996;Schrenzel et al, 1996;Bánfi et al, 1999;Bankers-Fulbright et al, 2001;Cherny et al, 2001;DeCoursey et al, 2001a;DeCoursey et al, 2001b), the membrane potential determined by current clamp measurements indicated that depolarization following activation of NADPH oxidase was not sufficient to activate the proton channel (Gordienko et al, 1996;Tare et al, 1998;Bánfi et al, 1999). Eosinophil resting membrane potential has been reported to be between -60 and -80 mV (Gordienko et al, 1996;Tare et al, 1998).…”

“…The NADPH oxidase and its associated proton channel are often activated by the same stimuli (DeCoursey et al, 2000;DeCoursey et al, 2001b;Cherny et al, 2001). We have previously shown that superoxide production by PMAstimulated eosinophils is inhibited by the selective PKCδ Journal of Cell Science 116 (15) Fig.…”