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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 200 μM Zn2+. Superimposed outward currents (bottom) elicited by a series of command pulses to +100 mV (top) in the presence of 200 μM Zn2+ at pH 8; other solution constituents as for Fig. 3. Command pulses in 5-s intervals. Record shown is an average of five trials. Controls before Zn2+ exposure (average of three trials) and after (average of seven trials) are also shown. (B) Time course of the Zn2+ effect. Abscissa, time of trial; ordinate, peak outward current; holding potential, −60 mV.
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Figure 5: Effect of 200 μM Zn2+. Superimposed outward currents (bottom) elicited by a series of command pulses to +100 mV (top) in the presence of 200 μM Zn2+ at pH 8; other solution constituents as for Fig. 3. Command pulses in 5-s intervals. Record shown is an average of five trials. Controls before Zn2+ exposure (average of three trials) and after (average of seven trials) are also shown. (B) Time course of the Zn2+ effect. Abscissa, time of trial; ordinate, peak outward current; holding potential, −60 mV.

Mentions: As originally described in snail neurons, voltage-dependent H+ currents are inhibited in a readily reversible manner by 1 mM Cd2+, Zn2+, Ni2+, and other divalent ions (Thomas and Meech 1982; Byerly and Suen 1989; Mahaut-Smith 1989). In human neutrophils, low concentrations of Zn2+ reversibly inhibit both the arachidonate-activated (Henderson et al. 1987, 1988) and voltage-gated (100 μM Zn2+; DeCoursey and Cherny 1993) H+ pathways. Zn2+ also reversibly inhibited the gp91-phox–mediated pathway in CHO91 cells. Fig. 5 A shows the amplitude and time course of membrane currents before and after addition of 200 μM Zn2+ to the bathing solution. The outward current was significantly reduced in the presence of Zn2+ and showed partial recovery when examined after washing. Fig. 5 B shows the time course of the effect. A series of depolarizing command pulses was used to monitor the outward current. At the point shown, the normal bathing solution was replaced by one containing 200 μM Zn2+. The pronounced inhibition of the large time- and voltage-dependent outward current recovered rapidly once the perfusion solution was returned to the Zn2+-free control solution.


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 200 μM Zn2+. Superimposed outward currents (bottom) elicited by a series of command pulses to +100 mV (top) in the presence of 200 μM Zn2+ at pH 8; other solution constituents as for Fig. 3. Command pulses in 5-s intervals. Record shown is an average of five trials. Controls before Zn2+ exposure (average of three trials) and after (average of seven trials) are also shown. (B) Time course of the Zn2+ effect. Abscissa, time of trial; ordinate, peak outward current; holding potential, −60 mV.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Effect of 200 μM Zn2+. Superimposed outward currents (bottom) elicited by a series of command pulses to +100 mV (top) in the presence of 200 μM Zn2+ at pH 8; other solution constituents as for Fig. 3. Command pulses in 5-s intervals. Record shown is an average of five trials. Controls before Zn2+ exposure (average of three trials) and after (average of seven trials) are also shown. (B) Time course of the Zn2+ effect. Abscissa, time of trial; ordinate, peak outward current; holding potential, −60 mV.
Mentions: As originally described in snail neurons, voltage-dependent H+ currents are inhibited in a readily reversible manner by 1 mM Cd2+, Zn2+, Ni2+, and other divalent ions (Thomas and Meech 1982; Byerly and Suen 1989; Mahaut-Smith 1989). In human neutrophils, low concentrations of Zn2+ reversibly inhibit both the arachidonate-activated (Henderson et al. 1987, 1988) and voltage-gated (100 μM Zn2+; DeCoursey and Cherny 1993) H+ pathways. Zn2+ also reversibly inhibited the gp91-phox–mediated pathway in CHO91 cells. Fig. 5 A shows the amplitude and time course of membrane currents before and after addition of 200 μM Zn2+ to the bathing solution. The outward current was significantly reduced in the presence of Zn2+ and showed partial recovery when examined after washing. Fig. 5 B shows the time course of the effect. A series of depolarizing command pulses was used to monitor the outward current. At the point shown, the normal bathing solution was replaced by one containing 200 μM Zn2+. The pronounced inhibition of the large time- and voltage-dependent outward current recovered rapidly once the perfusion solution was returned to the Zn2+-free control solution.

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