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A novel H(+) conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease.

Bánfi B, Schrenzel J, Nüsse O, Lew DP, Ligeti E, Krause KH, Demaurex N - J. Exp. Med. (1999)

Bottom Line: In this study, we describe the presence of two different types of H(+) currents in human eosinophils.In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC).In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Geneva University Hospitals, CH-1211 Geneva 4, Switzerland.

ABSTRACT
Efficient mechanisms of H(+) ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase-in analogy with mitochondrial cytochromes-has an inherent H(+) channel activity remains uncertain: electrophysiological studies did not find altered H(+) currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H(+) currents in human eosinophils. The "classical" H(+) current had properties similar to previously described H(+) conductances and was present in CGD cells. In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H(+) extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.

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The inward H+ currents do not require oxidase activity. (A) Effect of DPI (top) and of oxygen depletion (bottom) on the electron (left) and proton currents (right traces). Pipette solutions contained 25 μM GTPγS, 8 mM NADPH, pH 7.6; bath pH 7.1. Oxygen was removed from the bath solution by a 4-h preincubation with glucose-oxidase (50 mU/ml) and catalase (2,000 U/ml). Traces are representative of ≥7 experiments. (B) Current–voltage relationship of the H+ currents, measured as in the legend to Fig. 2, under control conditions (▵, PO2 = 25.7 ± 0.3 kPa), in the presence of DPI (□), or under low oxygen conditions (•, PO2 < 1 kPa). Data are mean ± SEM of ≥7 experiments for each condition.
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Figure 5: The inward H+ currents do not require oxidase activity. (A) Effect of DPI (top) and of oxygen depletion (bottom) on the electron (left) and proton currents (right traces). Pipette solutions contained 25 μM GTPγS, 8 mM NADPH, pH 7.6; bath pH 7.1. Oxygen was removed from the bath solution by a 4-h preincubation with glucose-oxidase (50 mU/ml) and catalase (2,000 U/ml). Traces are representative of ≥7 experiments. (B) Current–voltage relationship of the H+ currents, measured as in the legend to Fig. 2, under control conditions (▵, PO2 = 25.7 ± 0.3 kPa), in the presence of DPI (□), or under low oxygen conditions (•, PO2 < 1 kPa). Data are mean ± SEM of ≥7 experiments for each condition.

Mentions: The whole cell patch clamp technique 47 was used to measure whole cell membrane currents and membrane potential, essentially as described 2630. Patch pipettes were pulled from borosilicate glass (1.5 mM OD; Clark Electromedical Instruments) using a Flaming Brown automatic pipette puller (Sutter Instruments). Pipettes were fired–polished and had resistance in the range of 3–12 MΩ; seal resistance was 5–50 GΩ. Patch recordings were performed using a Axopatch 200A amplifier (Axon Instruments), in the current clamp or voltage clamp mode. Values for whole cell resistance varied between 2.5 and 30 GΩ; mean access resistance was between 10 and 30 MΩ. Cell capacitance ranged from 1.8 to 3.0 pF. Data were low-pass filtered at 20 Hz through an eight-pole Bessel filter and digitized at 100 Hz on a 12-bit A/D converter (Acqui; Sicmu), which also provided the voltage pulses. In addition, all experiments were recorded at high frequency (44 kHz) on a DAT tape recorder (DTR 1801; Biologic). Leak currents were small compared with the studied currents, and were subtracted only to calculate the current–voltage relationship of the time-dependent currents. Traces shown are not corrected for leak current and were smoothed by averaging 5 or 40 consecutive data points. In ∼30% of the cells, a transient (<10 s) outward current (presumably carried by K+) developed immediately after break-in (e.g., see Fig. 5 A, bottom left trace); this current was observed in both control and CGD cells.


A novel H(+) conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease.

Bánfi B, Schrenzel J, Nüsse O, Lew DP, Ligeti E, Krause KH, Demaurex N - J. Exp. Med. (1999)

The inward H+ currents do not require oxidase activity. (A) Effect of DPI (top) and of oxygen depletion (bottom) on the electron (left) and proton currents (right traces). Pipette solutions contained 25 μM GTPγS, 8 mM NADPH, pH 7.6; bath pH 7.1. Oxygen was removed from the bath solution by a 4-h preincubation with glucose-oxidase (50 mU/ml) and catalase (2,000 U/ml). Traces are representative of ≥7 experiments. (B) Current–voltage relationship of the H+ currents, measured as in the legend to Fig. 2, under control conditions (▵, PO2 = 25.7 ± 0.3 kPa), in the presence of DPI (□), or under low oxygen conditions (•, PO2 < 1 kPa). Data are mean ± SEM of ≥7 experiments for each condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195580&req=5

Figure 5: The inward H+ currents do not require oxidase activity. (A) Effect of DPI (top) and of oxygen depletion (bottom) on the electron (left) and proton currents (right traces). Pipette solutions contained 25 μM GTPγS, 8 mM NADPH, pH 7.6; bath pH 7.1. Oxygen was removed from the bath solution by a 4-h preincubation with glucose-oxidase (50 mU/ml) and catalase (2,000 U/ml). Traces are representative of ≥7 experiments. (B) Current–voltage relationship of the H+ currents, measured as in the legend to Fig. 2, under control conditions (▵, PO2 = 25.7 ± 0.3 kPa), in the presence of DPI (□), or under low oxygen conditions (•, PO2 < 1 kPa). Data are mean ± SEM of ≥7 experiments for each condition.
Mentions: The whole cell patch clamp technique 47 was used to measure whole cell membrane currents and membrane potential, essentially as described 2630. Patch pipettes were pulled from borosilicate glass (1.5 mM OD; Clark Electromedical Instruments) using a Flaming Brown automatic pipette puller (Sutter Instruments). Pipettes were fired–polished and had resistance in the range of 3–12 MΩ; seal resistance was 5–50 GΩ. Patch recordings were performed using a Axopatch 200A amplifier (Axon Instruments), in the current clamp or voltage clamp mode. Values for whole cell resistance varied between 2.5 and 30 GΩ; mean access resistance was between 10 and 30 MΩ. Cell capacitance ranged from 1.8 to 3.0 pF. Data were low-pass filtered at 20 Hz through an eight-pole Bessel filter and digitized at 100 Hz on a 12-bit A/D converter (Acqui; Sicmu), which also provided the voltage pulses. In addition, all experiments were recorded at high frequency (44 kHz) on a DAT tape recorder (DTR 1801; Biologic). Leak currents were small compared with the studied currents, and were subtracted only to calculate the current–voltage relationship of the time-dependent currents. Traces shown are not corrected for leak current and were smoothed by averaging 5 or 40 consecutive data points. In ∼30% of the cells, a transient (<10 s) outward current (presumably carried by K+) developed immediately after break-in (e.g., see Fig. 5 A, bottom left trace); this current was observed in both control and CGD cells.

Bottom Line: In this study, we describe the presence of two different types of H(+) currents in human eosinophils.In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC).In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Geneva University Hospitals, CH-1211 Geneva 4, Switzerland.

ABSTRACT
Efficient mechanisms of H(+) ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase-in analogy with mitochondrial cytochromes-has an inherent H(+) channel activity remains uncertain: electrophysiological studies did not find altered H(+) currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H(+) currents in human eosinophils. The "classical" H(+) current had properties similar to previously described H(+) conductances and was present in CGD cells. In contrast, the "novel" type of H(+) current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H(+) currents; and (e) it was approximately 20-fold more sensitive to Zn(2+) and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H(+) conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H(+) extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.

Show MeSH
Related in: MedlinePlus