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VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.

El Chemaly A, Okochi Y, Sasaki M, Arnaudeau S, Okamura Y, Demaurex N - J. Exp. Med. (2009)

Bottom Line: Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established.Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin.The migration defect was restored by the addition of a calcium ionophore.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Physiology and Metabolism, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
Neutrophils kill microbes with reactive oxygen species generated by the NADPH oxidase, an enzyme which moves electrons across membranes. Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established. We show that neutrophils from VSOP/Hv1-/- mice lack proton currents but have normal electron currents, indicating that these cells have a fully functional oxidase that cannot conduct protons. VSOP/Hv1-/- neutrophils had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice. Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin. Loss of VSOP/Hv1 also aborted calcium responses to chemoattractants, increased neutrophil spreading, and decreased neutrophil migration. The migration defect was restored by the addition of a calcium ionophore. Our findings indicate that proton channels extrude the acid and compensate the charge generated by the oxidase, thereby sustaining calcium entry signals that control the adhesion and motility of neutrophils. Loss of proton channels thus aborts superoxide production and causes a severe signaling defect in neutrophils.

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Electron currents, H2O2 production, and membrane potential of VSOP/Hv1−/− neutrophils. (A and B) Electron current recorded at −60 mV in a WT blood neutrophil in the perforated patch configuration. Currents were evoked by 100 nM PMA and blocked by 1 µM DPI. (B) Mean amplitude of the PMA-activated electron currents. Data are means ± SEM of 6 WT and 11 VSOP/Hv1−/− neutrophils from five WT and nine VSOP/Hv1−/− mice that were tested in 17 independent experiments. ns, not significant at P < 0.05 by an unpaired Student's t test. (C) Time-dependent H2O2 production in WT and VSOP/Hv1−/− blood neutrophils activated with PMA in the absence or presence of 1 mM of the proton channel blocker Zn2+ or 40 µg/ml of the proton-permeable channel gramicidin. Data are from two representative experiments that were independently performed more than three times. (D) Mean H2O2 production from WT and VSOP/Hv1−/− neutrophils. Data are means ± SD of three to five separate experiments done in triplicate from six WT and five VSOP/Hv1−/− mice. ***, P < 0.0001; *, P < 0.05, unpaired Student's t test. (E and F) Membrane potential changes measured with DiBAC4(3) during sequential addition of 1 µM PMA and 100 µM Zn2+ to blood neutrophils. (E) Mean responses of eight WT and nine VSOP/Hv1−/− neutrophils from four independent experiments. PMA evoked a larger depolarization in VSOP/Hv1−/− cells. Arrow indicates the direction of depolarization. (F) Mean change in DiBAC4(3) fluorescence evoked by PMA and PMA+Zn2+. Data are means ± SEM of eight WT and nine VSOP/Hv1−/− blood neutrophils from four independent experiments using five WT and four VSOP/Hv1−/− mice. *, P < 0.05, unpaired Student's t test.
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fig3: Electron currents, H2O2 production, and membrane potential of VSOP/Hv1−/− neutrophils. (A and B) Electron current recorded at −60 mV in a WT blood neutrophil in the perforated patch configuration. Currents were evoked by 100 nM PMA and blocked by 1 µM DPI. (B) Mean amplitude of the PMA-activated electron currents. Data are means ± SEM of 6 WT and 11 VSOP/Hv1−/− neutrophils from five WT and nine VSOP/Hv1−/− mice that were tested in 17 independent experiments. ns, not significant at P < 0.05 by an unpaired Student's t test. (C) Time-dependent H2O2 production in WT and VSOP/Hv1−/− blood neutrophils activated with PMA in the absence or presence of 1 mM of the proton channel blocker Zn2+ or 40 µg/ml of the proton-permeable channel gramicidin. Data are from two representative experiments that were independently performed more than three times. (D) Mean H2O2 production from WT and VSOP/Hv1−/− neutrophils. Data are means ± SD of three to five separate experiments done in triplicate from six WT and five VSOP/Hv1−/− mice. ***, P < 0.0001; *, P < 0.05, unpaired Student's t test. (E and F) Membrane potential changes measured with DiBAC4(3) during sequential addition of 1 µM PMA and 100 µM Zn2+ to blood neutrophils. (E) Mean responses of eight WT and nine VSOP/Hv1−/− neutrophils from four independent experiments. PMA evoked a larger depolarization in VSOP/Hv1−/− cells. Arrow indicates the direction of depolarization. (F) Mean change in DiBAC4(3) fluorescence evoked by PMA and PMA+Zn2+. Data are means ± SEM of eight WT and nine VSOP/Hv1−/− blood neutrophils from four independent experiments using five WT and four VSOP/Hv1−/− mice. *, P < 0.05, unpaired Student's t test.

Mentions: The failure to see proton currents in neutrophils activated with PMA could reflect a failure of the patch-clamped cells to mount a functional oxidase at their plasma membrane. To ensure that this was not the case, we recorded the electron currents (Ie−) generated by the electrogenic activity of the enzyme (Schrenzel et al., 1998). The perforated patch configuration was used to avoid rundown of the current that could occur in whole cell mode. As shown in Fig. 3 A, addition of 0.1 µM PMA evoked within 1 min an inward current at −60 mV that was fully inhibited by the subsequent addition of 1 µM of the oxidase inhibitor diphenyliodonium (DPI), thus fulfilling the criteria for Ie−. Importantly, the amplitude of Ie− recorded from VSOP/Hv1−/− and WT neutrophils was identical (−2.1 ± 0.4 vs. −2.3 ± 0.7 pA, n = 11 and 6, respectively; Fig. 3 B) and approached the value of −2.6 ± 0.5 pA recorded in PMA-activated mouse granulocytes by Morgan et al. (2007). The normal Ie− amplitude in VSOP/Hv1−/− neutrophils indicates that electrons are transferred from cytosolic NADPH to extracellular oxygen at normal rates, implying that VSOP/Hv1−/− neutrophils have a fully functional oxidase. The absence of proton currents in these cells thus proves that the oxidase does not contain a proton channel.


VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.

El Chemaly A, Okochi Y, Sasaki M, Arnaudeau S, Okamura Y, Demaurex N - J. Exp. Med. (2009)

Electron currents, H2O2 production, and membrane potential of VSOP/Hv1−/− neutrophils. (A and B) Electron current recorded at −60 mV in a WT blood neutrophil in the perforated patch configuration. Currents were evoked by 100 nM PMA and blocked by 1 µM DPI. (B) Mean amplitude of the PMA-activated electron currents. Data are means ± SEM of 6 WT and 11 VSOP/Hv1−/− neutrophils from five WT and nine VSOP/Hv1−/− mice that were tested in 17 independent experiments. ns, not significant at P < 0.05 by an unpaired Student's t test. (C) Time-dependent H2O2 production in WT and VSOP/Hv1−/− blood neutrophils activated with PMA in the absence or presence of 1 mM of the proton channel blocker Zn2+ or 40 µg/ml of the proton-permeable channel gramicidin. Data are from two representative experiments that were independently performed more than three times. (D) Mean H2O2 production from WT and VSOP/Hv1−/− neutrophils. Data are means ± SD of three to five separate experiments done in triplicate from six WT and five VSOP/Hv1−/− mice. ***, P < 0.0001; *, P < 0.05, unpaired Student's t test. (E and F) Membrane potential changes measured with DiBAC4(3) during sequential addition of 1 µM PMA and 100 µM Zn2+ to blood neutrophils. (E) Mean responses of eight WT and nine VSOP/Hv1−/− neutrophils from four independent experiments. PMA evoked a larger depolarization in VSOP/Hv1−/− cells. Arrow indicates the direction of depolarization. (F) Mean change in DiBAC4(3) fluorescence evoked by PMA and PMA+Zn2+. Data are means ± SEM of eight WT and nine VSOP/Hv1−/− blood neutrophils from four independent experiments using five WT and four VSOP/Hv1−/− mice. *, P < 0.05, unpaired Student's t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2812533&req=5

fig3: Electron currents, H2O2 production, and membrane potential of VSOP/Hv1−/− neutrophils. (A and B) Electron current recorded at −60 mV in a WT blood neutrophil in the perforated patch configuration. Currents were evoked by 100 nM PMA and blocked by 1 µM DPI. (B) Mean amplitude of the PMA-activated electron currents. Data are means ± SEM of 6 WT and 11 VSOP/Hv1−/− neutrophils from five WT and nine VSOP/Hv1−/− mice that were tested in 17 independent experiments. ns, not significant at P < 0.05 by an unpaired Student's t test. (C) Time-dependent H2O2 production in WT and VSOP/Hv1−/− blood neutrophils activated with PMA in the absence or presence of 1 mM of the proton channel blocker Zn2+ or 40 µg/ml of the proton-permeable channel gramicidin. Data are from two representative experiments that were independently performed more than three times. (D) Mean H2O2 production from WT and VSOP/Hv1−/− neutrophils. Data are means ± SD of three to five separate experiments done in triplicate from six WT and five VSOP/Hv1−/− mice. ***, P < 0.0001; *, P < 0.05, unpaired Student's t test. (E and F) Membrane potential changes measured with DiBAC4(3) during sequential addition of 1 µM PMA and 100 µM Zn2+ to blood neutrophils. (E) Mean responses of eight WT and nine VSOP/Hv1−/− neutrophils from four independent experiments. PMA evoked a larger depolarization in VSOP/Hv1−/− cells. Arrow indicates the direction of depolarization. (F) Mean change in DiBAC4(3) fluorescence evoked by PMA and PMA+Zn2+. Data are means ± SEM of eight WT and nine VSOP/Hv1−/− blood neutrophils from four independent experiments using five WT and four VSOP/Hv1−/− mice. *, P < 0.05, unpaired Student's t test.
Mentions: The failure to see proton currents in neutrophils activated with PMA could reflect a failure of the patch-clamped cells to mount a functional oxidase at their plasma membrane. To ensure that this was not the case, we recorded the electron currents (Ie−) generated by the electrogenic activity of the enzyme (Schrenzel et al., 1998). The perforated patch configuration was used to avoid rundown of the current that could occur in whole cell mode. As shown in Fig. 3 A, addition of 0.1 µM PMA evoked within 1 min an inward current at −60 mV that was fully inhibited by the subsequent addition of 1 µM of the oxidase inhibitor diphenyliodonium (DPI), thus fulfilling the criteria for Ie−. Importantly, the amplitude of Ie− recorded from VSOP/Hv1−/− and WT neutrophils was identical (−2.1 ± 0.4 vs. −2.3 ± 0.7 pA, n = 11 and 6, respectively; Fig. 3 B) and approached the value of −2.6 ± 0.5 pA recorded in PMA-activated mouse granulocytes by Morgan et al. (2007). The normal Ie− amplitude in VSOP/Hv1−/− neutrophils indicates that electrons are transferred from cytosolic NADPH to extracellular oxygen at normal rates, implying that VSOP/Hv1−/− neutrophils have a fully functional oxidase. The absence of proton currents in these cells thus proves that the oxidase does not contain a proton channel.

Bottom Line: Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established.Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin.The migration defect was restored by the addition of a calcium ionophore.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Physiology and Metabolism, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
Neutrophils kill microbes with reactive oxygen species generated by the NADPH oxidase, an enzyme which moves electrons across membranes. Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established. We show that neutrophils from VSOP/Hv1-/- mice lack proton currents but have normal electron currents, indicating that these cells have a fully functional oxidase that cannot conduct protons. VSOP/Hv1-/- neutrophils had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice. Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin. Loss of VSOP/Hv1 also aborted calcium responses to chemoattractants, increased neutrophil spreading, and decreased neutrophil migration. The migration defect was restored by the addition of a calcium ionophore. Our findings indicate that proton channels extrude the acid and compensate the charge generated by the oxidase, thereby sustaining calcium entry signals that control the adhesion and motility of neutrophils. Loss of proton channels thus aborts superoxide production and causes a severe signaling defect in neutrophils.

Show MeSH
Related in: MedlinePlus