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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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Equilibration of a pipette containing Lucifer yellow with cell cytoplasm. (A) Confocal images (Kalman average, n = 3) of CHO91-expressing cell collected before (image 1) and at 60-s (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 500 μM Lucifer yellow, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.5 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 cell contents at different time intervals after perforation of the cell membrane. (C). Confocal images within the focal plane of the cell (top) and pipette (bottom). Average fluorescence intensity of cell, 136 U; average fluorescence intensity of pipette, 133 U. Images shown are at three different positions for both cell and pipette. 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 1: Equilibration of a pipette containing Lucifer yellow with cell cytoplasm. (A) Confocal images (Kalman average, n = 3) of CHO91-expressing cell collected before (image 1) and at 60-s (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 500 μM Lucifer yellow, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.5 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 cell contents at different time intervals after perforation of the cell membrane. (C). Confocal images within the focal plane of the cell (top) and pipette (bottom). Average fluorescence intensity of cell, 136 U; average fluorescence intensity of pipette, 133 U. Images shown are at three different positions for both cell and pipette. 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: The time course of the exchange between the contents of a small cell and the contents of a patch pipette in the whole cell configuration has been studied both experimentally (Pusch and Neher 1988) and theoretically (Oliva et al. 1988; Mathias et al. 1990). If the contents of both pipette and cell remain homogenous, exchange at the pipette tip is rate limiting. Other influential factors are the cell volume and the size of the diffusing molecule (see materials and methods). In Fig. 1, a patch pipette filled with Lucifer yellow was used to record from a 25-μm-diameter CHO91 cell in the whole cell configuration. Although the intensity of the cytoplasm was undetectable 60 s after perforation of the cell membrane, fluorescence increased rapidly over the first 10 min (Fig. 1 A). Measurement of the average fluorescence within the boundary of the cell membrane showed an increase that followed an approximately exponential time course with a time constant of 150 s (Fig. 1 B). This is consistent with the findings of Pusch and Neher 1988, who found that molecules the size of Lucifer yellow (∼600 D) should transfer though a 5-MΩ patch pipette to the cytoplasm of a 25-μm-diameter cell with a time constant of between 85 and 169 s. A comparison between the maximum intensity of the cell (mean 136, n = 3) and the fluorescence of the pipette (mean 133, n = 3) showed that the dye was evenly distributed between cell and pipette (Fig. 1 C). There was no change in cytoplasmic fluorescence intensity if the cell membrane remained intact (not shown).


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)

Equilibration of a pipette containing Lucifer yellow with cell cytoplasm. (A) Confocal images (Kalman average, n = 3) of CHO91-expressing cell collected before (image 1) and at 60-s (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 500 μM Lucifer yellow, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.5 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 cell contents at different time intervals after perforation of the cell membrane. (C). Confocal images within the focal plane of the cell (top) and pipette (bottom). Average fluorescence intensity of cell, 136 U; average fluorescence intensity of pipette, 133 U. Images shown are at three different positions for both cell and pipette. 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

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getmorefigures.php?uid=PMC2230652&req=5

Figure 1: Equilibration of a pipette containing Lucifer yellow with cell cytoplasm. (A) Confocal images (Kalman average, n = 3) of CHO91-expressing cell collected before (image 1) and at 60-s (images 2–10), 2-min (images 11–15), and 5-min (images 16–18) intervals after onset of whole cell recording. Pipette solution: 500 μM Lucifer yellow, 119 mM TMA hydroxide, 3.7 mM EGTA, 0.74 mM CaCl2 adjusted to pH 6.5 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 cell contents at different time intervals after perforation of the cell membrane. (C). Confocal images within the focal plane of the cell (top) and pipette (bottom). Average fluorescence intensity of cell, 136 U; average fluorescence intensity of pipette, 133 U. Images shown are at three different positions for both cell and pipette. 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: The time course of the exchange between the contents of a small cell and the contents of a patch pipette in the whole cell configuration has been studied both experimentally (Pusch and Neher 1988) and theoretically (Oliva et al. 1988; Mathias et al. 1990). If the contents of both pipette and cell remain homogenous, exchange at the pipette tip is rate limiting. Other influential factors are the cell volume and the size of the diffusing molecule (see materials and methods). In Fig. 1, a patch pipette filled with Lucifer yellow was used to record from a 25-μm-diameter CHO91 cell in the whole cell configuration. Although the intensity of the cytoplasm was undetectable 60 s after perforation of the cell membrane, fluorescence increased rapidly over the first 10 min (Fig. 1 A). Measurement of the average fluorescence within the boundary of the cell membrane showed an increase that followed an approximately exponential time course with a time constant of 150 s (Fig. 1 B). This is consistent with the findings of Pusch and Neher 1988, who found that molecules the size of Lucifer yellow (∼600 D) should transfer though a 5-MΩ patch pipette to the cytoplasm of a 25-μm-diameter cell with a time constant of between 85 and 169 s. A comparison between the maximum intensity of the cell (mean 136, n = 3) and the fluorescence of the pipette (mean 133, n = 3) showed that the dye was evenly distributed between cell and pipette (Fig. 1 C). There was no change in cytoplasmic fluorescence intensity if the cell membrane remained intact (not shown).

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