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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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Effect of 120 mM pH buffer on intracellular pH. (A) Confocal images (Kalman average, n = 3) of CHO91 cell collected before (image 1) and at 60-second (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 50 μM BCECF, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.2 with Mes so that its final concentration was ∼120 mM. External solution: 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM EPPS, pH 8. Holding potential, −60 mV; bath temperature, 21–23°C; cell diameter, 25 μm. (B) Average florescence intensity of the cell contents at different time intervals after perforation of the cell membrane. (C) Confocal images (Kalman average, n = 3) within the focal plane of the cell (left) and pipette (right), obtained 50 min after achieving whole cell configuration. Average fluorescence intensity of six cells, 162; average fluorescence intensity of six pipettes, pH 6.2, 91. (D). Fluorescence intensity of BCECF calibrated by acquiring images of patch pipettes filled with 50 μM BCECF buffered to pH 6.1, 6.5, and 7.0. Abscissa, pH of pipette solution; ordinate, average pipette fluorescence intensity. A pseudo-color scale in which high intensity is denoted by red through orange, yellow, and green to blue, which corresponds to low fluorescence, is used to represent the fluorescence intensity in the images.
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Figure 2: Effect of 120 mM pH buffer on intracellular pH. (A) Confocal images (Kalman average, n = 3) of CHO91 cell collected before (image 1) and at 60-second (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 50 μM BCECF, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.2 with Mes so that its final concentration was ∼120 mM. External solution: 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM EPPS, pH 8. Holding potential, −60 mV; bath temperature, 21–23°C; cell diameter, 25 μm. (B) Average florescence intensity of the cell contents at different time intervals after perforation of the cell membrane. (C) Confocal images (Kalman average, n = 3) within the focal plane of the cell (left) and pipette (right), obtained 50 min after achieving whole cell configuration. Average fluorescence intensity of six cells, 162; average fluorescence intensity of six pipettes, pH 6.2, 91. (D). Fluorescence intensity of BCECF calibrated by acquiring images of patch pipettes filled with 50 μM BCECF buffered to pH 6.1, 6.5, and 7.0. Abscissa, pH of pipette solution; ordinate, average pipette fluorescence intensity. A pseudo-color scale in which high intensity is denoted by red through orange, yellow, and green to blue, which corresponds to low fluorescence, is used to represent the fluorescence intensity in the images.

Mentions: To examine the time course of the change in pHi, we used patch pipettes filled with the pH indicator BCECF buffered with 120 mM pH buffer (Mes). The molecular weight of BCECF (520) is similar to Lucifer yellow, while Mes (213 D) is somewhat smaller and should equilibrate with the cell more rapidly than the dye. The emitted fluorescence intensity of BCECF decreases with decreasing pH and so the cytoplasm should register only a small overall increase in fluorescence if its pH followed that of the pipette solution (pH 6.2). Fig. 2A and Fig. B, shows that the time course of the fluorescence increase was similar to that for Lucifer yellow, fluorescence reaching a maximum in 15–20 min. However, unlike Lucifer yellow, the fluorescence intensity of the cell and the pipette were not equal once the system had reached a steady state (Fig. 2 C) and remained different even after 60 min in the whole-cell configuration. The average fluorescence intensity of six cells (after 20 min in the whole cell configuration) was 163 U compared with 91 U for the pipette contents. Assuming that BCECF, like Lucifer yellow, was equally distributed between cell and pipette, and also that there was no interaction between BCECF and the cytoplasm, the inequality of fluorescence must arise from a difference in pH between the pipette solution and the cell contents. From the calibration curve (Fig. 2 D), the average value for pHi was near 6.9. As this was significantly different to the pipette solution (buffered to pH 6.2), it seemed that pHi was largely determined by endogenous cellular regulation. Nevertheless, the level of acidification achieved by using pipettes filled with 120 mM pH buffer was sufficient for the purposes of the present experiments. In control cells loaded with BCECF, pHi at rest is estimated to be 7.2 (Henderson et al., 1995).


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)

Effect of 120 mM pH buffer on intracellular pH. (A) Confocal images (Kalman average, n = 3) of CHO91 cell collected before (image 1) and at 60-second (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 50 μM BCECF, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.2 with Mes so that its final concentration was ∼120 mM. External solution: 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM EPPS, pH 8. Holding potential, −60 mV; bath temperature, 21–23°C; cell diameter, 25 μm. (B) Average florescence intensity of the cell contents at different time intervals after perforation of the cell membrane. (C) Confocal images (Kalman average, n = 3) within the focal plane of the cell (left) and pipette (right), obtained 50 min after achieving whole cell configuration. Average fluorescence intensity of six cells, 162; average fluorescence intensity of six pipettes, pH 6.2, 91. (D). Fluorescence intensity of BCECF calibrated by acquiring images of patch pipettes filled with 50 μM BCECF buffered to pH 6.1, 6.5, and 7.0. Abscissa, pH of pipette solution; ordinate, average pipette fluorescence intensity. A pseudo-color scale in which high intensity is denoted by red through orange, yellow, and green to blue, which corresponds to low fluorescence, is used to represent the fluorescence intensity in the images.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Effect of 120 mM pH buffer on intracellular pH. (A) Confocal images (Kalman average, n = 3) of CHO91 cell collected before (image 1) and at 60-second (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 50 μM BCECF, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.2 with Mes so that its final concentration was ∼120 mM. External solution: 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM EPPS, pH 8. Holding potential, −60 mV; bath temperature, 21–23°C; cell diameter, 25 μm. (B) Average florescence intensity of the cell contents at different time intervals after perforation of the cell membrane. (C) Confocal images (Kalman average, n = 3) within the focal plane of the cell (left) and pipette (right), obtained 50 min after achieving whole cell configuration. Average fluorescence intensity of six cells, 162; average fluorescence intensity of six pipettes, pH 6.2, 91. (D). Fluorescence intensity of BCECF calibrated by acquiring images of patch pipettes filled with 50 μM BCECF buffered to pH 6.1, 6.5, and 7.0. Abscissa, pH of pipette solution; ordinate, average pipette fluorescence intensity. A pseudo-color scale in which high intensity is denoted by red through orange, yellow, and green to blue, which corresponds to low fluorescence, is used to represent the fluorescence intensity in the images.
Mentions: To examine the time course of the change in pHi, we used patch pipettes filled with the pH indicator BCECF buffered with 120 mM pH buffer (Mes). The molecular weight of BCECF (520) is similar to Lucifer yellow, while Mes (213 D) is somewhat smaller and should equilibrate with the cell more rapidly than the dye. The emitted fluorescence intensity of BCECF decreases with decreasing pH and so the cytoplasm should register only a small overall increase in fluorescence if its pH followed that of the pipette solution (pH 6.2). Fig. 2A and Fig. B, shows that the time course of the fluorescence increase was similar to that for Lucifer yellow, fluorescence reaching a maximum in 15–20 min. However, unlike Lucifer yellow, the fluorescence intensity of the cell and the pipette were not equal once the system had reached a steady state (Fig. 2 C) and remained different even after 60 min in the whole-cell configuration. The average fluorescence intensity of six cells (after 20 min in the whole cell configuration) was 163 U compared with 91 U for the pipette contents. Assuming that BCECF, like Lucifer yellow, was equally distributed between cell and pipette, and also that there was no interaction between BCECF and the cytoplasm, the inequality of fluorescence must arise from a difference in pH between the pipette solution and the cell contents. From the calibration curve (Fig. 2 D), the average value for pHi was near 6.9. As this was significantly different to the pipette solution (buffered to pH 6.2), it seemed that pHi was largely determined by endogenous cellular regulation. Nevertheless, the level of acidification achieved by using pipettes filled with 120 mM pH buffer was sufficient for the purposes of the present experiments. In control cells loaded with BCECF, pHi at rest is estimated to be 7.2 (Henderson et al., 1995).

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
Related in: MedlinePlus