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Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H(+) pathway.

Henderson LM, Meech RW - J. Gen. Physiol. (1999)

Bottom Line: Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents.Stefani, and F.Bezanilla. 1997.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom BS8 1TD. l.m.henderson@bristol.ac.uk

ABSTRACT
Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H(+) conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O(2).(2)). Suspensions of CHO91 cells exhibit arachidonate-activatable H(+) fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909-5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H(+). As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199-216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590-1598), a lowered external pH (pH(o)) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 microM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10-20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2'7' bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pH(i)) was approximately 6.9, as though pH(i) was largely determined by endogenous cellular regulation. Arachidonate (20 microM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH(2)-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H(+) permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H(+) selectivity. Mechanisms of H(+) permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319-1327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.

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Expression of mutated gp91-phox in CHO-N and CHO-N3Leu cells. Confocal images of Cd2+-induced CHO-N cells (A and B) and CHO-N3Leu cells (D and E), immunostained with anti-hemagglutinin antibody. The levels of staining are similar in each case. The annular pattern of fluorescence was not observed in uninduced cells used as a control (C and F). Cell diameter, 20–25 μm. The fluorescence intensity is represented by a grey scale, with white being high and black low.
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Figure 8: Expression of mutated gp91-phox in CHO-N and CHO-N3Leu cells. Confocal images of Cd2+-induced CHO-N cells (A and B) and CHO-N3Leu cells (D and E), immunostained with anti-hemagglutinin antibody. The levels of staining are similar in each case. The annular pattern of fluorescence was not observed in uninduced cells used as a control (C and F). Cell diameter, 20–25 μm. The fluorescence intensity is represented by a grey scale, with white being high and black low.

Mentions: From the fluorescence intensity of immunostained cells, we were able to compare levels of expression in different mutated forms of gp91-phox. The cDNA constructs have three tandem repeats of the hemagglutinin epitope attached to their COOH-terminal ends. In Fig. 8, confocal images of Cd2+ induced CHO-N (A and B) and CHO-N3Leu (D and E) cells immunostained with antihemaglutinin antibody gave an annular pattern of fluorescence that was not observed in uninduced cells (C and F). This pattern of staining implies that the antigen is not only expressed, but that it is located at or in the plasma membrane and it is similar to that already observed in CHO cells expressing full-length gp91-phox (Henderson et al. 1995). The levels of staining do not differ greatly; expression in CHO-N3Leu being slightly greater than that in CHO-N.


Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H(+) pathway.

Henderson LM, Meech RW - J. Gen. Physiol. (1999)

Expression of mutated gp91-phox in CHO-N and CHO-N3Leu cells. Confocal images of Cd2+-induced CHO-N cells (A and B) and CHO-N3Leu cells (D and E), immunostained with anti-hemagglutinin antibody. The levels of staining are similar in each case. The annular pattern of fluorescence was not observed in uninduced cells used as a control (C and F). Cell diameter, 20–25 μm. The fluorescence intensity is represented by a grey scale, with white being high and black low.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230652&req=5

Figure 8: Expression of mutated gp91-phox in CHO-N and CHO-N3Leu cells. Confocal images of Cd2+-induced CHO-N cells (A and B) and CHO-N3Leu cells (D and E), immunostained with anti-hemagglutinin antibody. The levels of staining are similar in each case. The annular pattern of fluorescence was not observed in uninduced cells used as a control (C and F). Cell diameter, 20–25 μm. The fluorescence intensity is represented by a grey scale, with white being high and black low.
Mentions: From the fluorescence intensity of immunostained cells, we were able to compare levels of expression in different mutated forms of gp91-phox. The cDNA constructs have three tandem repeats of the hemagglutinin epitope attached to their COOH-terminal ends. In Fig. 8, confocal images of Cd2+ induced CHO-N (A and B) and CHO-N3Leu (D and E) cells immunostained with antihemaglutinin antibody gave an annular pattern of fluorescence that was not observed in uninduced cells (C and F). This pattern of staining implies that the antigen is not only expressed, but that it is located at or in the plasma membrane and it is similar to that already observed in CHO cells expressing full-length gp91-phox (Henderson et al. 1995). The levels of staining do not differ greatly; expression in CHO-N3Leu being slightly greater than that in CHO-N.

Bottom Line: Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents.Stefani, and F.Bezanilla. 1997.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom BS8 1TD. l.m.henderson@bristol.ac.uk

ABSTRACT
Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H(+) conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O(2).(2)). Suspensions of CHO91 cells exhibit arachidonate-activatable H(+) fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909-5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H(+). As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199-216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590-1598), a lowered external pH (pH(o)) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 microM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10-20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2'7' bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pH(i)) was approximately 6.9, as though pH(i) was largely determined by endogenous cellular regulation. Arachidonate (20 microM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl(-) concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH(2)-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H(+) permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H(+) selectivity. Mechanisms of H(+) permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319-1327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.

Show MeSH