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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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Calcium handling in VSOP/Hv1−/− neutrophils. Changes in cytosolic Ca2+ were measured with fura-2. (A) SOCE in blood neutrophils exposed to PMA for 20 min to activate the oxidase. Cells were deprived of Ca2+, treated with 1 µM thapsigargin to deplete Ca2+ stores, and exposed to 2 mM Ca2+ to reveal SOCE. Traces are means of 9 WT and 14 VSOP/Hv1−/− recordings (>10 cells each) from five WT and three VSOP/Hv1−/− mice. (B and C) Calcium elevations evoked by 10 µM fMIVIL in bone marrow neutrophils pretreated or not with PMA. Traces in B are means of 19 and 64 cells measured in five and seven independent experiments from four WT and four VSOP/Hv1−/− mice. Traces in C are means of four separate recordings (>10 cells each) from two WT and two VSOP/Hv1−/− mice. The chemotactic peptide was added at t = 0 (arrows). (D) Mean changes in cytosolic Ca2+ evoked by fMIVIL (area under the curve, AUC) and by Ca2+ readmission (Δ ratio amplitude). Data are means ± SD of the experiments in A–C. **, P < 0.001; *, P < 0.05, unpaired Student's t test.
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fig4: Calcium handling in VSOP/Hv1−/− neutrophils. Changes in cytosolic Ca2+ were measured with fura-2. (A) SOCE in blood neutrophils exposed to PMA for 20 min to activate the oxidase. Cells were deprived of Ca2+, treated with 1 µM thapsigargin to deplete Ca2+ stores, and exposed to 2 mM Ca2+ to reveal SOCE. Traces are means of 9 WT and 14 VSOP/Hv1−/− recordings (>10 cells each) from five WT and three VSOP/Hv1−/− mice. (B and C) Calcium elevations evoked by 10 µM fMIVIL in bone marrow neutrophils pretreated or not with PMA. Traces in B are means of 19 and 64 cells measured in five and seven independent experiments from four WT and four VSOP/Hv1−/− mice. Traces in C are means of four separate recordings (>10 cells each) from two WT and two VSOP/Hv1−/− mice. The chemotactic peptide was added at t = 0 (arrows). (D) Mean changes in cytosolic Ca2+ evoked by fMIVIL (area under the curve, AUC) and by Ca2+ readmission (Δ ratio amplitude). Data are means ± SD of the experiments in A–C. **, P < 0.001; *, P < 0.05, unpaired Student's t test.

Mentions: Besides decreasing superoxide production, the increased depolarization reduces the driving force for the entry of cations into cells. The lack of VSOP/Hv1 thus likely impacts on the influx of Ca2+, an ion which controls numerous cellular functions. In neutrophils, Ca2+ entry occurs predominantly across store-operated Ca2+ channels (SOCEs) activated by the depletion of intracellular Ca2+ stores (Scharff and Foder, 1993). To test whether VSOP/Hv1 ablation could have an impact on SOCE, we measured the changes in cytosolic Ca2+ concentration with fura-2. Neutrophils were exposed to PMA for 15 min to maximally activate the oxidase and depolarize the plasma membrane, thapsigargin was added in the absence of Ca2+ to deplete intracellular calcium stores, and Ca2+ was then readmitted to evoke SOCE. As shown in Fig. 4 A, thapsigargin evoked similar responses in control and VSOP/Hv1−/− neutrophils, indicating that Ca2+ release from stores is not altered by the loss of VSOP/Hv1 proton channels. However, SOCE was strongly reduced in VSOP/Hv1−/− neutrophils, as expected from the higher membrane voltage of these cells (Fig. 4, A and D). To see whether the blunted SOCE could alter Ca2+ responses to physiological agonists, we exposed cells to the bacterial peptide N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL), a potent agonist of the mouse formyl peptide receptors (Southgate et al., 2008). In the absence of preactivation with PMA, fMIVIL evoked biphasic Ca2+ elevations, reflecting an initial component of Ca2+ release followed by a delayed component of Ca2+ influx, as in human neutrophils (Demaurex et al., 1992). The integrated Ca2+ responses were significantly reduced in VSOP/Hv1−/− neutrophils exposed to fMIVIL for 30 min (Fig. 4, B and D), mainly because of a reduction in the delayed influx component of the Ca2+ signal. Interestingly, the fMIVIL Ca2+ responses were monophasic in neutrophils preactivated with PMA, suggesting that Ca2+ influx activates more rapidly in neutrophils exposed to the phorbol ester, possibly because of the phosphorylation of Ca2+ handling proteins. Remarkably, the Ca2+ responses were almost abrogated in VSOP/Hv1−/− neutrophils preactivated with PMA (Fig. 4, C and D). This indicates that VSOP/Hv1−/− neutrophils cannot mount effective Ca2+ signals when their oxidase is active, most likely as a result of the decreased SOCE imparted by the enhanced depolarization.


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)

Calcium handling in VSOP/Hv1−/− neutrophils. Changes in cytosolic Ca2+ were measured with fura-2. (A) SOCE in blood neutrophils exposed to PMA for 20 min to activate the oxidase. Cells were deprived of Ca2+, treated with 1 µM thapsigargin to deplete Ca2+ stores, and exposed to 2 mM Ca2+ to reveal SOCE. Traces are means of 9 WT and 14 VSOP/Hv1−/− recordings (>10 cells each) from five WT and three VSOP/Hv1−/− mice. (B and C) Calcium elevations evoked by 10 µM fMIVIL in bone marrow neutrophils pretreated or not with PMA. Traces in B are means of 19 and 64 cells measured in five and seven independent experiments from four WT and four VSOP/Hv1−/− mice. Traces in C are means of four separate recordings (>10 cells each) from two WT and two VSOP/Hv1−/− mice. The chemotactic peptide was added at t = 0 (arrows). (D) Mean changes in cytosolic Ca2+ evoked by fMIVIL (area under the curve, AUC) and by Ca2+ readmission (Δ ratio amplitude). Data are means ± SD of the experiments in A–C. **, P < 0.001; *, P < 0.05, unpaired Student's t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812533&req=5

fig4: Calcium handling in VSOP/Hv1−/− neutrophils. Changes in cytosolic Ca2+ were measured with fura-2. (A) SOCE in blood neutrophils exposed to PMA for 20 min to activate the oxidase. Cells were deprived of Ca2+, treated with 1 µM thapsigargin to deplete Ca2+ stores, and exposed to 2 mM Ca2+ to reveal SOCE. Traces are means of 9 WT and 14 VSOP/Hv1−/− recordings (>10 cells each) from five WT and three VSOP/Hv1−/− mice. (B and C) Calcium elevations evoked by 10 µM fMIVIL in bone marrow neutrophils pretreated or not with PMA. Traces in B are means of 19 and 64 cells measured in five and seven independent experiments from four WT and four VSOP/Hv1−/− mice. Traces in C are means of four separate recordings (>10 cells each) from two WT and two VSOP/Hv1−/− mice. The chemotactic peptide was added at t = 0 (arrows). (D) Mean changes in cytosolic Ca2+ evoked by fMIVIL (area under the curve, AUC) and by Ca2+ readmission (Δ ratio amplitude). Data are means ± SD of the experiments in A–C. **, P < 0.001; *, P < 0.05, unpaired Student's t test.
Mentions: Besides decreasing superoxide production, the increased depolarization reduces the driving force for the entry of cations into cells. The lack of VSOP/Hv1 thus likely impacts on the influx of Ca2+, an ion which controls numerous cellular functions. In neutrophils, Ca2+ entry occurs predominantly across store-operated Ca2+ channels (SOCEs) activated by the depletion of intracellular Ca2+ stores (Scharff and Foder, 1993). To test whether VSOP/Hv1 ablation could have an impact on SOCE, we measured the changes in cytosolic Ca2+ concentration with fura-2. Neutrophils were exposed to PMA for 15 min to maximally activate the oxidase and depolarize the plasma membrane, thapsigargin was added in the absence of Ca2+ to deplete intracellular calcium stores, and Ca2+ was then readmitted to evoke SOCE. As shown in Fig. 4 A, thapsigargin evoked similar responses in control and VSOP/Hv1−/− neutrophils, indicating that Ca2+ release from stores is not altered by the loss of VSOP/Hv1 proton channels. However, SOCE was strongly reduced in VSOP/Hv1−/− neutrophils, as expected from the higher membrane voltage of these cells (Fig. 4, A and D). To see whether the blunted SOCE could alter Ca2+ responses to physiological agonists, we exposed cells to the bacterial peptide N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL), a potent agonist of the mouse formyl peptide receptors (Southgate et al., 2008). In the absence of preactivation with PMA, fMIVIL evoked biphasic Ca2+ elevations, reflecting an initial component of Ca2+ release followed by a delayed component of Ca2+ influx, as in human neutrophils (Demaurex et al., 1992). The integrated Ca2+ responses were significantly reduced in VSOP/Hv1−/− neutrophils exposed to fMIVIL for 30 min (Fig. 4, B and D), mainly because of a reduction in the delayed influx component of the Ca2+ signal. Interestingly, the fMIVIL Ca2+ responses were monophasic in neutrophils preactivated with PMA, suggesting that Ca2+ influx activates more rapidly in neutrophils exposed to the phorbol ester, possibly because of the phosphorylation of Ca2+ handling proteins. Remarkably, the Ca2+ responses were almost abrogated in VSOP/Hv1−/− neutrophils preactivated with PMA (Fig. 4, C and D). This indicates that VSOP/Hv1−/− neutrophils cannot mount effective Ca2+ signals when their oxidase is active, most likely as a result of the decreased SOCE imparted by the enhanced depolarization.

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