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Thomas E DeCoursey

Publications and source records attributed to Thomas E DeCoursey.

15 recordsLinked to original sources
PubMed2006

Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2 alpha activity.

The prevailing hypothesis that a signalling pathway involving cPLA(2)alpha is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA(2)alpha as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (I(e)) and enhanced proton channel gating identically in the presence or absence of three specific cPLA(2)alpha inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF(3) (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA(2)alpha gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA(2)alpha is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised.

Animals↗
PubMed2006

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-PLA(2) or the production of superoxide anion (O(2*)(-)). 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 Zn(2+) inhibition of NADPH oxidase activity assessed by H(2)O(2) production, thus validating previous studies showing that Zn(2+) inhibited O(2*)(-) 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.

Animals↗
PubMed2006

Charge compensation during the phagocyte respiratory burst.

The phagocyte NADPH oxidase produces superoxide anion (O(2)(.-)) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O(2)(*-) to H(2)O(2) and then to HOCl. A small contribution by K+ or Cl- fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.

Animals↗
PubMed2005

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 pH(o) and pH(i) 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 pH(i), 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 (pH(o) = pH(i)) 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 pH(o) and pH(i) independently revealed that this pH dependence was entirely due to effects of pH(i) and that the oxidase is insensitive to pH(o). Surprisingly, the electron current in inside-out patches of membrane was only weakly sensitive to pH(i), 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.

Adult↗
PubMed2004

Voltage-gated proton channels help regulate pHi in rat alveolar epithelium.

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pH(i)) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO(3)(-)-free solutions [at external pH (pH(o)) 7.4]. Cells were acid-loaded by the NH(4)(+) prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 microM ZnCl(2), a proton channel inhibitor, slowed recovery about twofold. In addition, the pH(i) minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl(2) was observed at pH(o) 7.4 and pH(o) 8.0 and in normal and high-K(+) Ringer solutions. The observed rate of Zn(2+)-sensitive pH(i) recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pH(i) recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl(2) had no effect on pH(i) in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pH(i) recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.

Acids↗
PubMed2004

During the respiratory burst, do phagocytes need proton channels or potassium channels, or both?

The NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase enzyme complex, a crucial component of innate immunity, produces superoxide anion (O2-), which is a precursor to many reactive oxygen species. NADPH oxidase produces O2- by transferring electrons from intracellular NADPH across the membrane to extracellular (or phagosomal) oxygen and is thus electrogenic. It is widely believed that electroneutrality is preserved by proton flux through voltage-gated proton channels. A series of recent papers have challenged several key aspects of this view of the "respiratory burst." The most recent study solidifies the proposal that O2- and other reactive oxygen species produced by phagocytes are not toxic to microbes under physiological conditions. Further, an essential role for high-conductance, Ca2+-activated K+ (maxi-K+) channels in microbe killing is proposed. Finally, the results cast doubt on the widely held view that H+ efflux through voltage-gated proton channels (i) is the main mechanism of charge compensation, and (ii) is essential to continuous O2- production by the NADPH oxidase. My analysis of the new data and of a large body of data in the literature indicates that the proposed role of maxi-K+ channels in the respiratory burst is not yet credibly established. H+ efflux through proton channels thus remains the most viable mechanism for charge compensation and continuous O2- production. The important question of the toxicity of reactive oxygen species in phagocytes and in other cells, which has long been simply taken for granted, is a widespread assumption that deserves critical study.

Animals↗
PubMed2003

Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport.

Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2-. Electron flux is electrogenic, and rapidly depolarizes the membrane potential. Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux. Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.

Animals↗
PubMed2003

Diversity of voltage gated proton channels.

Voltage gated proton channels were first discovered in snail neurons and recently have been found in many mammalian cells. As their name suggests, H+ channels are sensitive to voltage, with an open probability that increases with membrane depolarization. Many properties that are shared by voltage-gated proton channels make them unique among ion channels. They show high selectivity for protons, strongly pH dependent gating, and a tiny single channel conductance. Although they are inhibited by divalent cations, including zinc and cadmium, no effective blockers exist. There is sufficient evidence to suggest that they are not water filled pores, unlike many other membrane bound ion channels. Instead, protons probably are conducted by a "hydrogen bonded chain" mechanism that resembles the Grotthuss mechanism in water. Differences in activation and deactivation kinetics of H+ currents in different cells suggest that there may be at least 4 isoforms of voltage gated proton channels. Gating kinetics may reflect specific functions. Voltage gated proton channels are well suited to extrude acid from cells and also may function in the extrusion of metabolic acid in the form of CO2 from the lungs. The best established function of H+ channels is in mammalian phagocytes, where they extrude protons to compensate for the charge separation created by the movement of electrons across the membrane by the bactericidal enzyme NADPH oxidase.

Animals↗
PubMed2003

Temperature dependence of NADPH oxidase in human eosinophils.

The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O2-. This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, Ie. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of Ie in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to >37 degrees C. For comparison, NADPH oxidase activity was assessed by cytochrome c reduction. The intrinsic temperature dependence of the assembled, functioning NADPH oxidase complex measured during rapid temperature increases to 37 degrees C was surprisingly weak: the Arrhenius activation energy Ea was only 14 kcal mol(-1) (Q10, 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20-30 degrees C, with Ea 25.1 kcal mol(-1) (Q10, 4.2). The maximum Ie measured at 34 degrees C was -30.5 pA. Above 30 degrees C, the temperature dependence of both Ie and O2- production was less pronounced. Above 37 degrees C, Ie was inhibited reversibly. After rapid temperature increases, a secondary increase in Ie ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37 degrees C than at 20 degrees C. Thus, the higher Q10 of steady-state Ie reflects both increased activity of each NADPH oxidase complex and preferential assembly of NADPH oxidase complexes at high temperature. In summary, NADPH oxidase activity in intact human eosinophils is maximal precisely at 37 degrees C.

Adult↗
PubMed2003

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

The enzyme NADPH oxidase in phagocytes is important in the body's 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 (I(e)) 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 I(e) 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 I(e), 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.

Animals↗
PubMed2003

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 < or = 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-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, sigmaH2. We analyze sigmaH2 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 gH 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 gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi < or = 5.5 near Vthreshold (the voltage at which gH turns on), single channel-like current events were observed with amplitudes 7-16 fA.

Adult↗
PubMed2002

Voltage-activated proton currents in human lymphocytes.

Voltage-activated proton currents are reported for the first time in human peripheral blood T and B lymphocytes and in the human leukaemic T cell line Jurkat E6-1. The properties of H(+) currents studied using tight-seal voltage-clamp recording techniques were similar in all cells. Changing the pH gradient by one unit caused a 47 mV shift in the reversal potential, demonstrating high selectivity of the channels for protons. H(+) current activation upon membrane depolarisation had a sigmoidal time course that could be fitted by a single exponential function after a brief delay. Increasing pH(o) shifted the activation threshold to more negative potentials, and increased both the H(+) current amplitude and the rate of activation. In lymphocytes studied at pH(i) 6.0, the activation threshold was more negative and the H(+) current density was three times larger than at pH(i) 7.0. Increasing the intracellular Ca(2+) concentration to 1 microM did not change H(+) current amplitude or kinetics detectably. Extracellularly applied Zn(2+) and Cd(2+) inhibited proton currents, slowing activation and shifting the voltage-activation curve to more positive potentials. The H(+) current amplitude was 100 times larger in CD19+ B lymphocytes and in Jurkat E6-1 cells than in CD3+ T lymphocytes. Following stimulation with the phorbol ester PMA, the H(+) current density in peripheral blood T lymphocytes and Jurkat T cells increased. In contrast, the H(+) current density of phorbol ester (PMA)-stimulated B lymphocytes was reduced and activation became slower. The pattern of expression of H(+) channels in lymphocytes appears well suited to their proposed role of charge compensation during the respiratory burst.

B-Lymphocytes↗
PubMed2002

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

Animals↗