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Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

Morgan D, Cherny VV, Price MO, Dinauer MC, DeCoursey TE - J. Gen. Physiol. (2002)

Bottom Line: Biol.PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells.Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1750 W Harrison, Chicago, IL 60612, USA.

ABSTRACT
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

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7D5 antibody staining of COSphox cells compared with COSWT cells. Cells harvested by brief trypsinization were incubated with 7D5 monoclonal antibody, which recognizes an extracellular epitope of gp91phox. Following incubation with a FITC-conjugated goat anti–mouse secondary antibody, cells were analyzed by flow cytometry. The marker M1 was set to exclude 99% of the COSWT cells (open histogram), and indicates the range defined as positively stained. By this criterion, 98.3% of the COSphox cells (solid histogram) were positively stained for gp91phox expression.
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fig5: 7D5 antibody staining of COSphox cells compared with COSWT cells. Cells harvested by brief trypsinization were incubated with 7D5 monoclonal antibody, which recognizes an extracellular epitope of gp91phox. Following incubation with a FITC-conjugated goat anti–mouse secondary antibody, cells were analyzed by flow cytometry. The marker M1 was set to exclude 99% of the COSWT cells (open histogram), and indicates the range defined as positively stained. By this criterion, 98.3% of the COSphox cells (solid histogram) were positively stained for gp91phox expression.

Mentions: To assess the efficiency of gp91phox expression in COSphox cells, we used the 7D5 antibody, which is directed against an extracellular epitope of the gp91phox molecule (Nakamura et al., 1988; Yu et al., 1998). Expression was tested in freshly thawed cells and passaged cells, in COSWT and COSphox cells, and with the CD-117 antibody as an isotype control. Fig. 5 illustrates histograms of the fluorescence intensity in COSWT (open histogram) and COSphox cells (solid histogram). 98% of the freshly thawed COSphox cells (Fig. 5) and 96% of COSphox cells maintained in culture for several weeks (unpublished data), as during the course of these experiments, stained positively for the 7D5 antibody. If cells with very low fluorescence (which presumably are dead cells) are included, then the 7D5-positive fraction is 95% of the freshly thawed COSphox cells and 93% of COSphox cells maintained in culture. Thus, the vast majority of COSphox cells express gp91phox at the plasma membrane.


Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components.

Morgan D, Cherny VV, Price MO, Dinauer MC, DeCoursey TE - J. Gen. Physiol. (2002)

7D5 antibody staining of COSphox cells compared with COSWT cells. Cells harvested by brief trypsinization were incubated with 7D5 monoclonal antibody, which recognizes an extracellular epitope of gp91phox. Following incubation with a FITC-conjugated goat anti–mouse secondary antibody, cells were analyzed by flow cytometry. The marker M1 was set to exclude 99% of the COSWT cells (open histogram), and indicates the range defined as positively stained. By this criterion, 98.3% of the COSphox cells (solid histogram) were positively stained for gp91phox expression.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233867&req=5

fig5: 7D5 antibody staining of COSphox cells compared with COSWT cells. Cells harvested by brief trypsinization were incubated with 7D5 monoclonal antibody, which recognizes an extracellular epitope of gp91phox. Following incubation with a FITC-conjugated goat anti–mouse secondary antibody, cells were analyzed by flow cytometry. The marker M1 was set to exclude 99% of the COSWT cells (open histogram), and indicates the range defined as positively stained. By this criterion, 98.3% of the COSphox cells (solid histogram) were positively stained for gp91phox expression.
Mentions: To assess the efficiency of gp91phox expression in COSphox cells, we used the 7D5 antibody, which is directed against an extracellular epitope of the gp91phox molecule (Nakamura et al., 1988; Yu et al., 1998). Expression was tested in freshly thawed cells and passaged cells, in COSWT and COSphox cells, and with the CD-117 antibody as an isotype control. Fig. 5 illustrates histograms of the fluorescence intensity in COSWT (open histogram) and COSphox cells (solid histogram). 98% of the freshly thawed COSphox cells (Fig. 5) and 96% of COSphox cells maintained in culture for several weeks (unpublished data), as during the course of these experiments, stained positively for the 7D5 antibody. If cells with very low fluorescence (which presumably are dead cells) are included, then the 7D5-positive fraction is 95% of the freshly thawed COSphox cells and 93% of COSphox cells maintained in culture. Thus, the vast majority of COSphox cells express gp91phox at the plasma membrane.

Bottom Line: Biol.PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells.Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Luke's Medical Center, 1750 W Harrison, Chicago, IL 60612, USA.

ABSTRACT
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

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