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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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Activation and deactivation of the currents in control and CGD cells. pHi 7.1 (A, B) or 7.6 (C, D), pHo 7.1. (A) Kinetics of current activation in control, CGD, and DEPC-treated cells. Currents elicited by a 5-s-long pulse from –20 mV to +60 mV were normalized to the maximal current recorded at this voltage and superimposed for comparison. (B) Voltage dependence of current activation. The time for half-maximal activation (t1/2 act) is plotted against the activating voltage. (C) Kinetics of current deactivation during repolarization from +60 mV to –20 mV. Cells were depolarized for various durations to induce currents of similar amplitude. Traces are representative of ≥9 experiments. (D) Voltage dependence of the deactivation time constants (τtail), estimated by fitting exponential curves to the currents measured after a pulse to +60 mV. The two fast kinetic components (τ1, ▪; τ2, •) are present in both control and CGD cells, whereas an additional third slow component (τ3, ▵) is absent from CGD cells. Data are mean ± SEM of ≥5 experiments for each condition.
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Figure 8: Activation and deactivation of the currents in control and CGD cells. pHi 7.1 (A, B) or 7.6 (C, D), pHo 7.1. (A) Kinetics of current activation in control, CGD, and DEPC-treated cells. Currents elicited by a 5-s-long pulse from –20 mV to +60 mV were normalized to the maximal current recorded at this voltage and superimposed for comparison. (B) Voltage dependence of current activation. The time for half-maximal activation (t1/2 act) is plotted against the activating voltage. (C) Kinetics of current deactivation during repolarization from +60 mV to –20 mV. Cells were depolarized for various durations to induce currents of similar amplitude. Traces are representative of ≥9 experiments. (D) Voltage dependence of the deactivation time constants (τtail), estimated by fitting exponential curves to the currents measured after a pulse to +60 mV. The two fast kinetic components (τ1, ▪; τ2, •) are present in both control and CGD cells, whereas an additional third slow component (τ3, ▵) is absent from CGD cells. Data are mean ± SEM of ≥5 experiments for each condition.

Mentions: To confirm the existence of two separate H+ conductive pathways, we analyzed in detail the kinetics of activation and deactivation of the currents. As shown in Fig. 8 A, superimposition of the currents elicited by a pulse to +60 mV and normalized to the peak current measured at this voltage revealed that current activation was more rapid in control than in CGD cells. Addition of DEPC, which had no effect on CGD cells (Fig. 7 D), slowed current activation to levels comparable to CGD cells (Fig. 8 A). At all voltages, the time for half-maximal activation (t1/2 act, measured by fitting a sigmoidal curve to the current) was significantly lower in control cells, and increased to values comparable to CGD cells upon addition of DEPC (Fig. 8 B).


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)

Activation and deactivation of the currents in control and CGD cells. pHi 7.1 (A, B) or 7.6 (C, D), pHo 7.1. (A) Kinetics of current activation in control, CGD, and DEPC-treated cells. Currents elicited by a 5-s-long pulse from –20 mV to +60 mV were normalized to the maximal current recorded at this voltage and superimposed for comparison. (B) Voltage dependence of current activation. The time for half-maximal activation (t1/2 act) is plotted against the activating voltage. (C) Kinetics of current deactivation during repolarization from +60 mV to –20 mV. Cells were depolarized for various durations to induce currents of similar amplitude. Traces are representative of ≥9 experiments. (D) Voltage dependence of the deactivation time constants (τtail), estimated by fitting exponential curves to the currents measured after a pulse to +60 mV. The two fast kinetic components (τ1, ▪; τ2, •) are present in both control and CGD cells, whereas an additional third slow component (τ3, ▵) is absent from CGD cells. Data are mean ± SEM of ≥5 experiments for each condition.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195580&req=5

Figure 8: Activation and deactivation of the currents in control and CGD cells. pHi 7.1 (A, B) or 7.6 (C, D), pHo 7.1. (A) Kinetics of current activation in control, CGD, and DEPC-treated cells. Currents elicited by a 5-s-long pulse from –20 mV to +60 mV were normalized to the maximal current recorded at this voltage and superimposed for comparison. (B) Voltage dependence of current activation. The time for half-maximal activation (t1/2 act) is plotted against the activating voltage. (C) Kinetics of current deactivation during repolarization from +60 mV to –20 mV. Cells were depolarized for various durations to induce currents of similar amplitude. Traces are representative of ≥9 experiments. (D) Voltage dependence of the deactivation time constants (τtail), estimated by fitting exponential curves to the currents measured after a pulse to +60 mV. The two fast kinetic components (τ1, ▪; τ2, •) are present in both control and CGD cells, whereas an additional third slow component (τ3, ▵) is absent from CGD cells. Data are mean ± SEM of ≥5 experiments for each condition.
Mentions: To confirm the existence of two separate H+ conductive pathways, we analyzed in detail the kinetics of activation and deactivation of the currents. As shown in Fig. 8 A, superimposition of the currents elicited by a pulse to +60 mV and normalized to the peak current measured at this voltage revealed that current activation was more rapid in control than in CGD cells. Addition of DEPC, which had no effect on CGD cells (Fig. 7 D), slowed current activation to levels comparable to CGD cells (Fig. 8 A). At all voltages, the time for half-maximal activation (t1/2 act, measured by fitting a sigmoidal curve to the current) was significantly lower in control cells, and increased to values comparable to CGD cells upon addition of DEPC (Fig. 8 B).

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