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. 2009 May 5;106(18):7642-7.
doi: 10.1073/pnas.0902761106. Epub 2009 Apr 16.

Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst

I Scott Ramsey  1 , Evelyne Ruchti, J Stefan Kaczmarek, David E Clapham
Affiliations

Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst

I Scott Ramsey et al. Proc Natl Acad Sci U S A. .

Abstract

Granulocytes generate a "respiratory burst" of NADPH oxidase-dependent superoxide anion (O(2)(-*)) production that is required for efficient clearance of bacterial pathogens. Hv1 mediates a voltage-gated H(+) channel activity that is proposed to serve a charge-balancing role in granulocytic phagocytes such as neutrophils and eosinophils. Using mice in which the gene encoding Hv1 is replaced by beta-Geo reporter protein sequence, we show that Hv1 expression is required for measurable voltage-gated H(+) current in unstimulated phagocytes. O(2)(-*) production is substantially reduced in the absence of Hv1, suggesting that Hv1 contributes a majority of the charge compensation required for optimal NADPH oxidase activity. Despite significant reduction in superoxide production, Hv1(-/-) mice are able to clear several types of bacterial infections.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation of Hv1−/− mice. RRN293 ES cells contain a Bay Genomics genetrap vector insertion in the Hvcn1 gene. The transgene serves as a surrogate splice acceptor for wt exon 2 that produces an in-frame chimeric mRNA encoding Hvcn1 exons 1 and 2 followed by β-Geo sequence. (A) Triplex genomic PCR using reverse primers specific for both Hvcn1 and β-Geo amplifies a ≈1.5-kb band from the wt allele (lane 1) and a ≈1.0-kb band from the transgenic allele (lane 3); both alleles are detected in heterozygous wt/RRN293 mice (lane 2). (B) Expression of Hv1 protein (32 kDa) is readily detected in spleen (lanes 1 and 2), BMC (lanes 3 and 4), and PBL (lanes 5 and 6) isolated from wt/wt mice (+/+, lanes 1, 3, and 5) but is absent in RRN293/RRN293 mouse tissue (−/−, lanes 2, 4, and 6).
Fig. 2.
Fig. 2.
RRN293/RRN293 leukocytes lack detectable voltage-gated H+ currents. (A and B) Representative whole-cell currents (Lower) resulting from the indicated voltage steps (Upper) in neutrophils (Lympholyte M) purified from wt/wt (+/+, Left) or RRN293/RRN293 (−/−, Right) B5 mouse blood. (Inset) The pH of solutions (TMA6.5 pipette, TMA7.5 bath) is shown. (C) Representative ISTEP vs. voltage relations for the cells shown in A under symmetrical [H+] conditions and after bath superfusion to impose a 1 pH unit outward [H+] gradient (○, +/+ TMA6.5 bath; ●, +/+ TMA7.5 bath; □, −/− TMA6.5 bath; ■, −/−TMA7.5 bath). (D) Whole-cell current (ISTEP, +60 mV) in granulocytes (Robbins PMN) purified from wt/wt (+/+) or RRN293/RRN293 (−/−) mouse blood (Na6.5 pipette and Na7.5 bath solutions). Solid line indicates the mean current level, and box indicates SEM (+/+, 58.6 ± 10.5 pA; −/−, 2.7 ± 0.4 pA; n = 14 cells per genotype).
Fig. 3.
Fig. 3.
Superoxide anion (O2−∙) production is decreased in Hv1−/− BMC. O2−∙ secretion was measured by using the cytochrome c reduction assay in intact BMC isolated from wt/wt (+/+, filled symbols) or RRN293/RRN293 (−/−, open symbols) B5 mice in the absence (black circles) or presence of 200 nM PMA (blue triangles) or 200 nM PMA + 10 μM DPI (red squares). (A) Time course of O2−∙ production after vehicle or PMA addition at t = 0 min in intact BMC. O2−∙ production after 10-min incubation (37 °C) in the presence of 200 nM PMA was 9.93 ± 1.24 nmol/106 cells (+/+) and 3.60 ± 0.18 nmol/106 cells (−/−); mean ± SEM, n = 4 mice per genotype). (B) The rates of PMA-stimulated O2−∙ production in intact BMC between 1 and 3 min after PMA addition were: +/+, 1.39 ± 0.23 nmol/min per 106 cells;−/−, 0.47 ± 0.04 nmol/min per 106 cells; mean ± SEM, n = 4 (solid lines). Data and symbols are as shown in A. (C) Time course of O2−∙ production after vehicle or AA (100 μM) addition at t = 0 min in a representative pair of wt/wt and RRN293/RRN293 littermate BMC sonicates assayed in parallel. (Inset) Time course of O2−∙ production after vehicle or PMA addition at t = 0 min in intact BMC before sonication. Data are expressed as nmol O2−∙/min per 106 cells. Open and filled symbols are as in A. (D) Western blot showing p22phox expression in lysates prepared from nontransfected COS-7, COS-phox, or HL-60 cells (lanes 1–3) or BMC isolated from 2 pairs of RRN293/RRN293 (−/−, lanes 5 and 6) or wt/wt (+/+, lanes 7 and 8) mouse littermates.
Fig. 4.
Fig. 4.
Hv1 deletion decreases bacterial clearance in vitro. (A) Mouse BMC (5 × 105) from wt/wt (solid columns, n = 8) or RRN293/RRN293 (hatched columns, n = 7) mice were incubated (30 min, 37 °C) with S. aureus in the absence (white columns) or presence of DPI (10 μM; gray columns) before addition of gentamicin and washing. Cell pellets were lysed in 1% saponin, and surviving bacteria were serially diluted and elaborated overnight on selective agar plates. (B) Data shown as the fraction of surviving CFU in the absence of DPI (control) divided by the CFU surviving the presence of DPI to isolate the DPI-sensitive component of bacterial clearance (wt/wt, white column; RRN293/RRN293, gray column). (C) Bacterial survival 24 h after i.p. inoculation of 1 × 108 CFU S. aureus in wt/wt (white column, n = 6) or RRN293/RRN293 (gray column, n = 8) mice. (D) Bacterial survival 6 h after i.p. inoculation of 5 × 109 CFU S. aureus in wt/wt (white column, n = 7) or RRN293/RRN293 (gray column, n = 7) B5 mice. Data represent mean values ± SEM; indicated P values are from Student's unpaired t test.
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