title: Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis.
abstract: Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH(i) in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na(+)/H(+) antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH(i) decreased sharply, and recovery required both Na(+)/H(+) antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn(2+) resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn(2+). Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA.
authors.forename: Deri
authors.initials: D
authors.lastname: Morgan
authors.forename: Melania
authors.initials: M
authors.lastname: Capasso
authors.forename: Boris
authors.initials: B
authors.lastname: Musset
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Cherny
authors.forename: Eduardo
authors.initials: E
authors.lastname: Ríos
authors.forename: Martin J S
authors.initials: MJ
authors.lastname: Dyer
authors.forename: Thomas E
authors.initials: TE
authors.lastname: DeCoursey
mesh_terms: D000328:Adult; D000818:Animals; D001578:Benzopyrans; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007473:Ion Channels / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism; D009284:Naphthols; D009504:Neutrophils / Q000502:physiology*; D010587:Phagocytosis / Q000502:physiology*; D011522:Protons; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
chemical_list: D001578:Benzopyrans; D005456:Fluorescent Dyes; C540876:Hv1 proton channel, mouse; D007473:Ion Channels; D009284:Naphthols; D011522:Protons; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; C091666:seminaphthorhodaminefluoride; D019255:NADPH Oxidases