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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.

Show MeSH
Effect of external pH on tail current amplitude and reversal potential. Depolarizing commands having elicited an outward current, the membrane was repolarized, and time-dependent tail currents were recorded. Current records from three different cells are shown in A–C. Capacitive transients (which lasted 1–2 ms) were blanked from each record. Pipette solutions as for Fig. 3. (A) External bathing solution was set at pH 8.0. Depolarizing command, 0 mV. During successive trials, the repolarizing command was in the range −40 to −100 mV. Currents are shown superimposed; average of four trials; holding potential −60 mV. (B) External bathing solution was set at pH 7.5. Depolarizing command, 80 mV; membrane repolarized in the range −40 to −100 mV. Superimposed traces from single trial; holding potential −60 mV. (C) External bathing solution was set at pH 7.0. Depolarizing command, 120 mV; membrane repolarized in the range 0 to −60 mV. Superimposed traces are an average of three trials; holding potential −60 mV. (D) Abscissa, membrane potential during repolarizing command; ordinate, tail current amplitude measured between the end of the capacitive transient (1–2 ms) and a steady level 200 ms later; bath solutions, pH 8.0 (□), 7.5 (○) and 7.0 (▵). Lines through data were drawn according to a constant field equation (Goldman 1943; Hodgkin and Katz 1949) with pHi taken as 6.9. H+ permeability chosen to fit pHo 8 data; pHo 7.5 and 7 data scaled up by factors of 3.5 and 7.5. Filled symbols represent data from other cells in the same external solutions: pH 8.0 (▪), scaling factor, 1; pH 7.5 (•), scaling factor, 3.5; pH 7.0 (▴), scaling factor, 11. Bath temperature 21–23°C.
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Figure 6: Effect of external pH on tail current amplitude and reversal potential. Depolarizing commands having elicited an outward current, the membrane was repolarized, and time-dependent tail currents were recorded. Current records from three different cells are shown in A–C. Capacitive transients (which lasted 1–2 ms) were blanked from each record. Pipette solutions as for Fig. 3. (A) External bathing solution was set at pH 8.0. Depolarizing command, 0 mV. During successive trials, the repolarizing command was in the range −40 to −100 mV. Currents are shown superimposed; average of four trials; holding potential −60 mV. (B) External bathing solution was set at pH 7.5. Depolarizing command, 80 mV; membrane repolarized in the range −40 to −100 mV. Superimposed traces from single trial; holding potential −60 mV. (C) External bathing solution was set at pH 7.0. Depolarizing command, 120 mV; membrane repolarized in the range 0 to −60 mV. Superimposed traces are an average of three trials; holding potential −60 mV. (D) Abscissa, membrane potential during repolarizing command; ordinate, tail current amplitude measured between the end of the capacitive transient (1–2 ms) and a steady level 200 ms later; bath solutions, pH 8.0 (□), 7.5 (○) and 7.0 (▵). Lines through data were drawn according to a constant field equation (Goldman 1943; Hodgkin and Katz 1949) with pHi taken as 6.9. H+ permeability chosen to fit pHo 8 data; pHo 7.5 and 7 data scaled up by factors of 3.5 and 7.5. Filled symbols represent data from other cells in the same external solutions: pH 8.0 (▪), scaling factor, 1; pH 7.5 (•), scaling factor, 3.5; pH 7.0 (▴), scaling factor, 11. Bath temperature 21–23°C.

Mentions: Small differences in the expression of gp91-phox may explain the observed variation in current amplitude, but there were also differences in time course. Fig. 3 C shows a cell in which the current steadily increases throughout each 800 ms command step, while in Fig. 6 A the currents rapidly achieve a steady level. In a population of cells with a steadily rising outward current, the mean amplitude after 800 ms at +80 mV was 3.4 nA (n = 9; SD 1.1 nA; range 1.7–4.6 nA); in a population of cells with steady outward currents the mean amplitude at +80 mV was 4.2 nA (n = 9; SD 2.1 nA; range 1.5–7.0 nA). Voltage-gated proton currents in other cell types show similar steadily increasing currents, but at present only a tentative explanation can be put forward to account for them; it is possible that during prolonged depolarizing commands negatively charged buffer molecules contribute to the pipette current by leaving the cytoplasm. The resulting acidification at the membrane near the pipette tip will produce a progressive, local shift in activation towards more negative membrane potentials. The currents were sustained during command pulses of 2 s and showed no inactivation (not shown), which is another characteristic of voltage-gated proton currents.


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 external pH on tail current amplitude and reversal potential. Depolarizing commands having elicited an outward current, the membrane was repolarized, and time-dependent tail currents were recorded. Current records from three different cells are shown in A–C. Capacitive transients (which lasted 1–2 ms) were blanked from each record. Pipette solutions as for Fig. 3. (A) External bathing solution was set at pH 8.0. Depolarizing command, 0 mV. During successive trials, the repolarizing command was in the range −40 to −100 mV. Currents are shown superimposed; average of four trials; holding potential −60 mV. (B) External bathing solution was set at pH 7.5. Depolarizing command, 80 mV; membrane repolarized in the range −40 to −100 mV. Superimposed traces from single trial; holding potential −60 mV. (C) External bathing solution was set at pH 7.0. Depolarizing command, 120 mV; membrane repolarized in the range 0 to −60 mV. Superimposed traces are an average of three trials; holding potential −60 mV. (D) Abscissa, membrane potential during repolarizing command; ordinate, tail current amplitude measured between the end of the capacitive transient (1–2 ms) and a steady level 200 ms later; bath solutions, pH 8.0 (□), 7.5 (○) and 7.0 (▵). Lines through data were drawn according to a constant field equation (Goldman 1943; Hodgkin and Katz 1949) with pHi taken as 6.9. H+ permeability chosen to fit pHo 8 data; pHo 7.5 and 7 data scaled up by factors of 3.5 and 7.5. Filled symbols represent data from other cells in the same external solutions: pH 8.0 (▪), scaling factor, 1; pH 7.5 (•), scaling factor, 3.5; pH 7.0 (▴), scaling factor, 11. Bath temperature 21–23°C.
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Related In: Results  -  Collection

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Figure 6: Effect of external pH on tail current amplitude and reversal potential. Depolarizing commands having elicited an outward current, the membrane was repolarized, and time-dependent tail currents were recorded. Current records from three different cells are shown in A–C. Capacitive transients (which lasted 1–2 ms) were blanked from each record. Pipette solutions as for Fig. 3. (A) External bathing solution was set at pH 8.0. Depolarizing command, 0 mV. During successive trials, the repolarizing command was in the range −40 to −100 mV. Currents are shown superimposed; average of four trials; holding potential −60 mV. (B) External bathing solution was set at pH 7.5. Depolarizing command, 80 mV; membrane repolarized in the range −40 to −100 mV. Superimposed traces from single trial; holding potential −60 mV. (C) External bathing solution was set at pH 7.0. Depolarizing command, 120 mV; membrane repolarized in the range 0 to −60 mV. Superimposed traces are an average of three trials; holding potential −60 mV. (D) Abscissa, membrane potential during repolarizing command; ordinate, tail current amplitude measured between the end of the capacitive transient (1–2 ms) and a steady level 200 ms later; bath solutions, pH 8.0 (□), 7.5 (○) and 7.0 (▵). Lines through data were drawn according to a constant field equation (Goldman 1943; Hodgkin and Katz 1949) with pHi taken as 6.9. H+ permeability chosen to fit pHo 8 data; pHo 7.5 and 7 data scaled up by factors of 3.5 and 7.5. Filled symbols represent data from other cells in the same external solutions: pH 8.0 (▪), scaling factor, 1; pH 7.5 (•), scaling factor, 3.5; pH 7.0 (▴), scaling factor, 11. Bath temperature 21–23°C.
Mentions: Small differences in the expression of gp91-phox may explain the observed variation in current amplitude, but there were also differences in time course. Fig. 3 C shows a cell in which the current steadily increases throughout each 800 ms command step, while in Fig. 6 A the currents rapidly achieve a steady level. In a population of cells with a steadily rising outward current, the mean amplitude after 800 ms at +80 mV was 3.4 nA (n = 9; SD 1.1 nA; range 1.7–4.6 nA); in a population of cells with steady outward currents the mean amplitude at +80 mV was 4.2 nA (n = 9; SD 2.1 nA; range 1.5–7.0 nA). Voltage-gated proton currents in other cell types show similar steadily increasing currents, but at present only a tentative explanation can be put forward to account for them; it is possible that during prolonged depolarizing commands negatively charged buffer molecules contribute to the pipette current by leaving the cytoplasm. The resulting acidification at the membrane near the pipette tip will produce a progressive, local shift in activation towards more negative membrane potentials. The currents were sustained during command pulses of 2 s and showed no inactivation (not shown), which is another characteristic of voltage-gated proton currents.

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