Vladimir V. Cherny

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

The enzyme NADPH oxidase in phagocytes is important in the bodys defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current–voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils

Indirect evidence indicates that a proton-selective conductance is activated during the respiratory burst in neutrophils. A voltage- and time-dependent H(+)-selective conductance, gH, in human neutrophils is demonstrated here directly by the whole-cell patch-clamp technique. The gH is extremely low at large negative potentials, increases slowly upon membrane depolarization, and does not inactivate. It is enhanced at high external pH or low internal pH and is inhibited by Cd2+ and Zn2+. Arachidonic acid, which plays a pivotal role in inflammatory reactions, amplifies the gH. The properties of the gH described here are compatible with its activation during the respiratory burst in stimulated neutrophils, in which it may facilitate sustained superoxide anion release by dissipating metabolically generated acid.

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

VOLTAGE-ACTIVATED HYDROGEN ION CURRENTS

Discovered in snail neurons by Thomas and Meech in 1982 [151], voltage-activated H+-selective currents have been found in an increasing number of species and cells, including human phagocytes and skeletal muscle. The properties of the H + currents are similar in all of these preparations. The H + conductance, g~, is undetectably small at large negative potentials, and activates in a time-dependent manner during depolarizing voltage pulses, apparently like other voltage-gated ion channels. The estimated single channel conductance is quite small, 1 0 fS, and thus a carrier mechanism cannot be formally excluded. The gH is highly selective, with no detectable permeability to other ions and a relative permeability PH/PN, > 106. Protons probably traverse the membrane by hopping across a hydrogenbonded chain within an integral membrane protein, although the molecule responsible is entirely unknown at present. The voltage dependence of H + channel gating is modulated by pH on both sides of the membrane such that only outward H + currents are observed at fixed membrane potentials. Activation of the gH would therefore alkalinize the cytoplasm in an intact cell. The fully activated gH alkalinizes small cells two orders of magnitude faster than other pH regulating transporters, at no metabolic cost to the cell. Voltage-activated H + channels may serve as a safety valve in situations of excessive metabolic acid production. The goal of this review is less to summarize the lit-

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

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 pHi 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, pHi 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 Zn2+ 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 Zn2+. 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.

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel.

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, HV1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, HV1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules ‘frozen’ in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of HV1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of HV1 requires an acidic group at the selectivity filter.

Interactions between NADPH oxidase‐related proton and electron currents in human eosinophils

1 Proton and electron currents in human eosinophils were studied using the permeabilized‐patch voltage‐clamp technique, with an applied NH4+ gradient to control pHi. 2 Voltage‐gated proton channels in unstimulated human eosinophils studied with the permeabilized‐patch approach had properties similar to those reported in whole‐cell studies. 3 Superoxide anion (O2−) release assessed by cytochrome c reduction was compared in human eosinophils and neutrophils stimulated by phorbol myristate acetate (PMA). PMA‐stimulated O2 release was more transient and the maximum rate was three times greater in eosinophils. 4 In PMA‐activated eosinophils, the H+ current amplitude (IH) at +60 mV increased 4.7‐fold, activation was 4.0 times faster, deactivation (tail current decay) was 5.4 times slower, the H+ conductance‐voltage (gH‐V) relationship was shifted ‐43 mV, and diphenylene iodinium (DPI)‐inhibitable inward current reflecting electron flow through NADPH oxidase was activated. The data reveal that PMA activates the H+ efflux during the respiratory burst by modulating the properties of H+ channels, not simply as a result of NADPH oxidase activity. 5 The electrophysiological response of eosinophils to PMA resembled that reported in human neutrophils, but PMA activated larger proton and electron currents in eosinophils and the response was more transient. 6 ZnCl2 slowed the activation of H+ currents and shifted the gH‐V relationship to more positive voltages. These effects occurred at similar ZnCl2 concentrations in eosinophils before and after PMA stimulation. These data are compatible with the existence of a single type of H+ channel in eosinophils that is modulated during the respiratory burst.

The pH dependence of NADPH oxidase in human eosinophils

NADPH oxidase generates reactive oxygen species that are essential to innate immunity against microbes. Like most enzymes, it is sensitive to pH, although the relative importance of pHo and pHi has not been clearly distinguished. We have taken advantage of the electrogenic nature of NADPH oxidase to determine its pH dependence in patch‐clamped individual human eosinophils using the electron current to indicate enzyme activity. Electron current stimulated by PMA (phorbol myristate acetate) was recorded in both perforated‐patch configuration, using an NH4+ gradient to control pHi, and in excised, inside‐out patches of membrane. No electron current was detected in cells or excised patches from eosinophils from a patient with chronic granulomatous disease. When the pH was varied symmetrically (pHo= pHi) in cells in perforated‐patch configuration, NADPH oxidase‐generated electron current was maximal at pH 7.5, decreasing drastically at higher or lower values. Varying pHo and pHi independently revealed that this pH dependence was entirely due to effects of pHi and that the oxidase is insensitive to pHo. Surprisingly, the electron current in inside‐out patches of membrane was only weakly sensitive to pHi, indicating that the enzyme turnover rate per se is not strongly pH dependent. The most likely interpretation is that assembly or deactivation of the NADPH oxidase complex has one or more pH‐sensitive steps, and that pH‐dependent changes in electron current in intact cells mainly reflect different numbers of active complexes at different pH.

The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2^{.}}^{-}\end{equation*}\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2^{.}}^{-}\end{equation*}\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.

Properties of Single Voltage-gated Proton Channels in Human Eosinophils Estimated by Noise Analysis and by Direct Measurement

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi ≤ 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, g H-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}_{{\mathrm{H}}}^{{\mathrm{2}}}\end{equation*}\end{document}. We analyze \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\sigma}}}_{{\mathrm{H}}}^{{\mathrm{2}}}\end{equation*}\end{document} data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic g H was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic g H, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi ≤ 5.5 near V threshold (the voltage at which g H turns on), single channel–like current events were observed with amplitudes 7–16 fA.