Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils

Abstract

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes. Phorbol myristate acetate (PMA) is a potent agonist for this ''respiratory burst'' in human neutrophils. Although stoichiometric H ؉ efflux occurs during the respiratory burst, efforts to stimulate voltagegated H ؉ channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H ؉ current properties, each of which increases the H ؉ current at any given voltage: (i) a 40-mV negative shift in the H ؉ conductance-voltage (gH-V) relationship; (ii) faster activation [smaller activation time constant ( act)] during depolarizing pulses; (iii) slower deactivation [larger deactivation time constant ( tail)] on repolarization; and (iv) a larger maximum H ؉ conductance (gH,max). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of tail with a time course paralleling the inhibition of electron current. However, the amplitudes of H ؉ and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H ؉ channels rather than inducing a new H ؉ channel.respiratory burst ͉ hydrogen ion ͉ pH ͉ proton channel ͉ ion channels N ADPH oxidase is a major bactericidal weapon of neutrophils and other phagocytes (1). On stimulation, the enzyme complex assembles in the membrane and produces superoxide anion, the precursor to several bactericidal reactive oxygen species. NADPH oxidase is electrogenic (2), releasing a proton into the cytoplasm for each superoxide anion released into the phagocytic vacuole or extracellular medium. As a result, pH changes reflecting increased H ϩ permeability are seen during the respiratory burst in phagocytes (2, 3) after stimulation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Voltagegated proton channels, discovered in snail neurons (4), were postulated to mediate the increased H ϩ permeability during the respiratory burst in human neutrophils (2, 5, 6). The importance of H ϩ efflux as a means of charge compensation is emphasized by the inhibition of PMA-stimulated superoxide anion production by Zn 2ϩ or Cd 2ϩ , which presumably act by inhibiting proton current (7,8). However, attempts to demonstrate effects of PMA on H ϩ currents in phagocytes studied in the whole-cell patchclamp configuration have heretofore been unsuccessful. It is therefore unclear whether the g H observed in phagoc...

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

“…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).…”

“…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.…”

Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?

“…Defects in any one of the enzyme subunits cause chronic granulomatous disease (CGD), a genetic disease characterized by severe and recurrent bacterial infections that were fatal in childhood before the advent of antibiotics (Stasia and Li, 2008). The oxidase is electrogenic (Henderson et al, 1987) as it translocates electrons across the plasma membrane in order to produce superoxide, and the electron currents can be measured with patch-clamp electrodes in cells with high plasma membrane oxidase activity, such as neutrophils and eosinophils (Schrenzel et al, 1998;DeCoursey et al, 2000). Unless compensated, the extrusion of the negatively charged electron will depolarize the phagocyte plasma membrane to extremely high voltages, and values of +60 mV and of up to +180 mV have been reported in intact cells (Jankowski and Grinstein, 1999) and in excised patches, respectively Petheo and Demaurex, 2005).…”

“…Proton currents of activated phagocytes have larger amplitude, faster activation kinetics (s act ), a À40 mV lower threshold of voltage activation, and markedly slower inactivation kinetics (s tail ). The last two effects are the most obvious and require the presence of a functional oxidase, because neutrophils from CGD patients only display a À13 mV shift in activation threshold and nearly normal channel closing kinetics when stimulated with phorbol 12-myristate 13-acetate (PMA) (DeCoursey et al, 2000(DeCoursey et al, , 2001Banfi et al, 1999;Petheo et al, 2003). Hv1 channels exogenously expressed in a B cell hybridoma cell line also display a À10 mV shift in activation threshold when phosphorylated, a response that requires threonine 29 ).…”