The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel

Abstract

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

Ligands of purinergic receptors stimulate electrogenic H+‐transport of neutrophils

“…As a compensating mechanism, the operation of a H ÷-transporting pathway has been suggested in the plasma membrane that could allow the efflux of protons in the direction of their electrochemical gradient [3,4]. The existence of an electrogenic H+-transporting pathway has been demonstrated in the plasma membrane of neutrophil granulocytes [3][4][5][6][7], macrophages [8] and HL-60 granulocytes [9], on the basis of intracellular pH-determinations by fluorescent dyes [3 6] and by whole cell patch clamp measurements [7][8][9].…”

Proton production, regulation and pathophysiological roles in the mammalian brain

“…HVCN1 has multiple functions in different cell types. For example, in neutrophils it mediates the positive charge compensation associated with oxidative bursts during phagocytosis [51,52] , while in tracheal epithelium it mediates acid secretion [49] . In addition, activation of HVCN1…”

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).…”

“…The activity of the oxidase was shown early on to require a compensating charge , which was immediately postulated to be provided by voltage-gated proton channels (Henderson et al, 1987) whose activity was then recorded electrically in phagocytes (Demaurex et al, 1993a,b;DeCoursey and Cherny, 1993). An alternative mechanism was postulated in 2004 by Tony Segal, who proposed that the large-conductance Ca 2+ -activated K + channel (BKCa) was responsible for charge compensation during respiratory burst and thus essential for innate immunity (Ahluwalia et al, 2004).…”

“…Recent studies from mice bearing a targeted disruption within the HVCN1 gene (Hv1 -/-) confirmed that Hv1 is the only proton channel expressed in phagocytes and invalidated definitively a longstanding hypothesis postulating that the oxidase is itself a proton channel (Henderson et al, 1987Henderson and Meech, 2002;Banfi et al, 2000;DeCoursey et al, 2002;Maturana et al, 2002). All attempts to record proton currents in Hv1-deficient phagocytes yielded absolutely flat traces, even after activation of cells with PMA (Ramsey et al, 2009;Morgan et al, 2009;El Chemaly et al, 2010;Okochi et al, 2009), a procedure that evoked electron currents of similar amplitude in Hv1 -/-and in wild-type (WT) Fig.…”