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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 20 μM sodium arachidonate. (A) Superimposed outward currents elicited by command pulses to +20, +60, +100 mV before (solid lines) and in the presence of (data points) 20 μM sodium arachidonate; other solution constituents as for Fig. 3. Scale bar refers to the test data; the control currents having been scaled up. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Command pulses in 20-s intervals. Holding potential, −60 mV; potential during tail current, −40 mV. (B) Voltage dependence of activation of the outward current. Abscissa, command potential; ordinate, normalized membrane conductance at end of 750-ms command pulse before (○) and in presence of (•) sodium arachidonate. Tail current reversal potential taken as −55 mV. The lines fitted to the experimental results were calculated from the equation: GH = GH,max/{1 + exp[(V1/2 − Vm)/20 mV]}, where GH,max is the maximum chord conductance and V1/2 is the potential at which GH is 0.5 GH,max. For sodium arachidonate, V1/2, −2 mV; GH,max, normalized to 1. For control, V1/2, 21 mV; GH,max, 0.83. (C) Inward current elicited from CHO91 cells by a series of command pulses to −20 mV in a bath solution of 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM HEPES, pH 6.8, recorded before (I), in the presence of 20 μM arachidonate (II), and after arachidonate (III). Currents were recorded immediately upon obtaining whole cell configuration, before equilibration of cytosol and pipette solution so that pHi was higher than pHo. Holding potential, −60 mV; command pulses in 5-s intervals; bath temperature, 21–23°C.
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Figure 4: Effect of 20 μM sodium arachidonate. (A) Superimposed outward currents elicited by command pulses to +20, +60, +100 mV before (solid lines) and in the presence of (data points) 20 μM sodium arachidonate; other solution constituents as for Fig. 3. Scale bar refers to the test data; the control currents having been scaled up. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Command pulses in 20-s intervals. Holding potential, −60 mV; potential during tail current, −40 mV. (B) Voltage dependence of activation of the outward current. Abscissa, command potential; ordinate, normalized membrane conductance at end of 750-ms command pulse before (○) and in presence of (•) sodium arachidonate. Tail current reversal potential taken as −55 mV. The lines fitted to the experimental results were calculated from the equation: GH = GH,max/{1 + exp[(V1/2 − Vm)/20 mV]}, where GH,max is the maximum chord conductance and V1/2 is the potential at which GH is 0.5 GH,max. For sodium arachidonate, V1/2, −2 mV; GH,max, normalized to 1. For control, V1/2, 21 mV; GH,max, 0.83. (C) Inward current elicited from CHO91 cells by a series of command pulses to −20 mV in a bath solution of 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM HEPES, pH 6.8, recorded before (I), in the presence of 20 μM arachidonate (II), and after arachidonate (III). Currents were recorded immediately upon obtaining whole cell configuration, before equilibration of cytosol and pipette solution so that pHi was higher than pHo. Holding potential, −60 mV; command pulses in 5-s intervals; bath temperature, 21–23°C.

Mentions: The H+ pathway associated with NADPH oxidase (Henderson and Chappell 1992), like that of gp91-phox (Henderson et al. 1995) and the voltage-gated H+ pathway in human neutrophils (DeCoursey and Cherny 1993), is activated by low levels of sodium arachidonate. Fig. 4 shows a similar effect on the voltage-gated currents observed in CHO91 cells. Currents elicited by control commands to 20, 40, and 100 mV are shown as solid lines (Fig. 4 A), whereas those recorded during the application of 20 μM sodium arachidonate are shown as data points. The scale on the right refers to the test data; the control currents having been increased to match. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Thus, although sodium arachidonate produced a significant increase in the amplitude of the voltage-gated currents, the time course of activation remained quite unaffected. The effect on current amplitude was rapidly reversible (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)

Effect of 20 μM sodium arachidonate. (A) Superimposed outward currents elicited by command pulses to +20, +60, +100 mV before (solid lines) and in the presence of (data points) 20 μM sodium arachidonate; other solution constituents as for Fig. 3. Scale bar refers to the test data; the control currents having been scaled up. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Command pulses in 20-s intervals. Holding potential, −60 mV; potential during tail current, −40 mV. (B) Voltage dependence of activation of the outward current. Abscissa, command potential; ordinate, normalized membrane conductance at end of 750-ms command pulse before (○) and in presence of (•) sodium arachidonate. Tail current reversal potential taken as −55 mV. The lines fitted to the experimental results were calculated from the equation: GH = GH,max/{1 + exp[(V1/2 − Vm)/20 mV]}, where GH,max is the maximum chord conductance and V1/2 is the potential at which GH is 0.5 GH,max. For sodium arachidonate, V1/2, −2 mV; GH,max, normalized to 1. For control, V1/2, 21 mV; GH,max, 0.83. (C) Inward current elicited from CHO91 cells by a series of command pulses to −20 mV in a bath solution of 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM HEPES, pH 6.8, recorded before (I), in the presence of 20 μM arachidonate (II), and after arachidonate (III). Currents were recorded immediately upon obtaining whole cell configuration, before equilibration of cytosol and pipette solution so that pHi was higher than pHo. Holding potential, −60 mV; command pulses in 5-s intervals; bath temperature, 21–23°C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230652&req=5

Figure 4: Effect of 20 μM sodium arachidonate. (A) Superimposed outward currents elicited by command pulses to +20, +60, +100 mV before (solid lines) and in the presence of (data points) 20 μM sodium arachidonate; other solution constituents as for Fig. 3. Scale bar refers to the test data; the control currents having been scaled up. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Command pulses in 20-s intervals. Holding potential, −60 mV; potential during tail current, −40 mV. (B) Voltage dependence of activation of the outward current. Abscissa, command potential; ordinate, normalized membrane conductance at end of 750-ms command pulse before (○) and in presence of (•) sodium arachidonate. Tail current reversal potential taken as −55 mV. The lines fitted to the experimental results were calculated from the equation: GH = GH,max/{1 + exp[(V1/2 − Vm)/20 mV]}, where GH,max is the maximum chord conductance and V1/2 is the potential at which GH is 0.5 GH,max. For sodium arachidonate, V1/2, −2 mV; GH,max, normalized to 1. For control, V1/2, 21 mV; GH,max, 0.83. (C) Inward current elicited from CHO91 cells by a series of command pulses to −20 mV in a bath solution of 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, 100 mM HEPES, pH 6.8, recorded before (I), in the presence of 20 μM arachidonate (II), and after arachidonate (III). Currents were recorded immediately upon obtaining whole cell configuration, before equilibration of cytosol and pipette solution so that pHi was higher than pHo. Holding potential, −60 mV; command pulses in 5-s intervals; bath temperature, 21–23°C.
Mentions: The H+ pathway associated with NADPH oxidase (Henderson and Chappell 1992), like that of gp91-phox (Henderson et al. 1995) and the voltage-gated H+ pathway in human neutrophils (DeCoursey and Cherny 1993), is activated by low levels of sodium arachidonate. Fig. 4 shows a similar effect on the voltage-gated currents observed in CHO91 cells. Currents elicited by control commands to 20, 40, and 100 mV are shown as solid lines (Fig. 4 A), whereas those recorded during the application of 20 μM sodium arachidonate are shown as data points. The scale on the right refers to the test data; the control currents having been increased to match. The scaling factors were 2 (20 mV), 1.24 (60 mV), and 1.4 (100 mV). Thus, although sodium arachidonate produced a significant increase in the amplitude of the voltage-gated currents, the time course of activation remained quite unaffected. The effect on current amplitude was rapidly reversible (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