The NADPH oxidase complex (NOX2) assembles in the phagosomal membrane upon activation to shuttles electrons from NADPH in the cytoplasm across the membrane to oxygen in the phagosomal lumen converting oxygen into the superoxide radical anion (O2.-) (Winterbourn CC et al. 2006). The large flux of electrons across the membrane-bound NOX2 complex together with H+ release during the oxidation and regeneration of NADPH in cytosol create a charge imbalance that depolarizes the membrane. To compensate the membrane depolarization NADPH oxidase activity is accompanied by activation of a voltage-gated proton (HV1) channel (Demaurex N & El Chemaly A 2010; El Chemaly A et al. 2010; Petheo GL et al. 2010; Kovacs I et al. 2014; Henderson LM et al. 1987, 1988; Nunes P et al. 2013). Proton channels extrude the cytosolic acid, repolarize the phagosomal membrane, and deliver cytosolic protons to the phagocytic vesicle lumen (Henderson LM et al. 1987, 1988; Morgan D et al. 2009; El Chemaly A et al. 2010).
The crucial function of voltage gated proton channels in compensating the electrogenic activity of NADPH oxidase during phagocytosis was demonstrated in human phagocytes (DeCoursey TE et al. 2000; Morgan D et al. 2009; Petheo GL et al. 2010; Kovacs I et al. 2014; Henderson LM et al. 1987, 1988). Hv1 knockout (KO) mice have been shown to lack detectable proton current in bone marrow or peripheral blood phagocytic cells (Morgan D et al. 2009; Ramsey IS et al. 2009; El Chemaly A et al. 2010; Capasso M et al. 2010). Furthermore, VSOP/Hv1-/- mouse cells had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice (Morgan D et al. 2009; El Chemaly A et al. 2010).
HV1 channels differentially regulate the phagosomal pH in neutrophils and macrophages. In macrophages, HV1 channels contributed to rapid phagosomal acidification together with V-ATPases, proton transporters, that are delivered to nascent phagosomes to generate a transmembrane pH gradient of >4 (El Chemaly A et al, 2014). In contrast, HV1 channels maintained a higher pH by sustaining high-level ROS production that is thought to inhibit V-ATPase accumulation on phagosomes in neutrophils (Jankowski A et al. 2002). In a 2015 study using a probe that is more sensitive at higher pH, an average pH closer to 9 was measured in individual phagosomes in neutrophils (Levine AP et al. 2015). The early alkalization of neutrophil phagosomes was also linked to proton consumption during the generation of hydrogen peroxide (Segal AW et al. 1981; Levine AP et al. 2015). Neutrophil phagosomes also exhibited a high proton leak, which was initiated upon activation of the NADPH oxidase, and this activation counteracted phagosomal acidification (Jankowski A et al. 2002).
