Activation of H+ conductance in neutrophils requires assembly of components of the respiratory burst oxidase but not its redox function.

Abstract

In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp9l-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H' translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H' extrusion. CGD cells expressing diminished amounts of cytochrome displayed H' conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+ . Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity. (J. Clin. Invest. 1994. 93:1770-1775

Arachidonic acid activatable electrogenic H+ transport in the absence of cytochrome b558 in human T lymphocytes

“…It is doubtful whether a protein highly specific for phagocytic cells could be responsible for the electrogenic H+-movements of embryologically so distinct cell types. Finally, our data are in good agreement with recent findings on CGD patients' neutrophils, in which a H + conductance activated by depolarisation and internal acidification has been detected despite of absence of gp91pho× [30].…”

“…T and B cells" H +-movements through the plasma membrane of T and B lymphocytes prepared from human tonsils were measured by monitoring intracellular pH changes with the same technique as used for the characterization of the electrogenic H +-transporting pathway of neutrophil granulocytes, peripheral lymphocytes, macrophages and enterocytes [4,7,11,16,23]. Briefly, intracellular pH was measured on the basis of the fluorescence of the pH indicator dye BCECF.…”

“…The molecular and functional relationship between the NADPH oxidase and the putative H+-channel was investigated in several studies. First, alteration of the electrogenic H+-transport was observed in neutrophils of CGD patients lacking different components of the NADPH oxidase [15,16]. In a previous study we showed that in peripheral pig lymphocytes, which possess an electrogenic H+-transporter in many aspects similar to that of granulocytes and macrophages, the two specific cytosolic components of the oxidase are not detectable.…”

The gp91 Component of NADPH Oxidase Is Not the Voltage-gated Proton Channel in Phagocytes, but It Helps

“…The failure to see H ϩ current at pH i 7.5 is especially surprising, because the currents at pH i 6.9 are an order of magnitude greater than in any mammalian cell. Finally, the outward currents in CHO cells activate anomalously rapidly, within Ͻ100 ms, whereas act for phagocyte H ϩ channels is typically seconds (9,17,24,27,29,31). It was reported recently that transient gp91 phox expression in COS-7 cells results in voltage-gated proton currents (15).…”

“…Several lines of evidence have suggested that the gp91 phox component of the NADPH oxidase complex might be the proton channel that is activated during the respiratory burst (2,3,7,8). The presence of H ϩ currents in monocytes from gp91 phox -deficient CGD 1 patients appeared to refute this idea (9), but Henderson and Chappell (10) argued that these data were inconclusive. Furthermore, it has been reported that heterologous expression of gp91 phox results in the appearance of proton fluxes or proton currents resembling those activated during the respiratory burst (8,(11)(12)(13)(14)(15).…”