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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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Ion currents recorded from CHO cells under whole cell voltage clamp. Superimposed currents recorded in response to a standard stepped-voltage protocol as indicated at the top of the figure; commands in the range −40 to 140 mV in 20-mV intervals. Pipette solution: 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°C. (A) Untransfected CHO cell, (B) uninduced (i.e., nonexpressing) CHO91 cell, (C) gp91-phox–expressing CHO91 cell.
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Figure 3: Ion currents recorded from CHO cells under whole cell voltage clamp. Superimposed currents recorded in response to a standard stepped-voltage protocol as indicated at the top of the figure; commands in the range −40 to 140 mV in 20-mV intervals. Pipette solution: 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°C. (A) Untransfected CHO cell, (B) uninduced (i.e., nonexpressing) CHO91 cell, (C) gp91-phox–expressing CHO91 cell.

Mentions: In previous experiments, suspensions of CHO91 cells have been used to demonstrate that gp91-phox functions as an H+ pathway when activated by sodium arachidonate (Henderson et al. 1995). To determine whether the gp91-phox pathway was voltage gated, we recorded membrane currents from CHO91 cells using the whole cell configuration of the patch-clamp technique. There are low levels of arachidonate-activated H+ conduction in both untransfected CHO cells and transfected but uninduced (i.e., nonexpressing) CHO91 cells (Henderson et al. 1995) but, in keeping with the report by Cherny et al. 1996, any voltage-gated H+ currents present were within the noise level of our recordings. Fig. 3 A shows data from a typical untransfected CHO cell bathed in pH 8.0 saline, under conditions designed to exclude currents other than those carried by H+. The cell was depolarized by a series of voltage commands, as shown in the protocol at top. In this and the four other untransfected cells tested, the whole-cell currents were all within the noise levels shown. Similar records were obtained for nonexpressing CHO91 cells under the same conditions (n = 6). Fig. 3 B shows a typical example.


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)

Ion currents recorded from CHO cells under whole cell voltage clamp. Superimposed currents recorded in response to a standard stepped-voltage protocol as indicated at the top of the figure; commands in the range −40 to 140 mV in 20-mV intervals. Pipette solution: 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°C. (A) Untransfected CHO cell, (B) uninduced (i.e., nonexpressing) CHO91 cell, (C) gp91-phox–expressing CHO91 cell.
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Related In: Results  -  Collection

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Figure 3: Ion currents recorded from CHO cells under whole cell voltage clamp. Superimposed currents recorded in response to a standard stepped-voltage protocol as indicated at the top of the figure; commands in the range −40 to 140 mV in 20-mV intervals. Pipette solution: 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°C. (A) Untransfected CHO cell, (B) uninduced (i.e., nonexpressing) CHO91 cell, (C) gp91-phox–expressing CHO91 cell.
Mentions: In previous experiments, suspensions of CHO91 cells have been used to demonstrate that gp91-phox functions as an H+ pathway when activated by sodium arachidonate (Henderson et al. 1995). To determine whether the gp91-phox pathway was voltage gated, we recorded membrane currents from CHO91 cells using the whole cell configuration of the patch-clamp technique. There are low levels of arachidonate-activated H+ conduction in both untransfected CHO cells and transfected but uninduced (i.e., nonexpressing) CHO91 cells (Henderson et al. 1995) but, in keeping with the report by Cherny et al. 1996, any voltage-gated H+ currents present were within the noise level of our recordings. Fig. 3 A shows data from a typical untransfected CHO cell bathed in pH 8.0 saline, under conditions designed to exclude currents other than those carried by H+. The cell was depolarized by a series of voltage commands, as shown in the protocol at top. In this and the four other untransfected cells tested, the whole-cell currents were all within the noise levels shown. Similar records were obtained for nonexpressing CHO91 cells under the same conditions (n = 6). Fig. 3 B shows a typical example.

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