Correspondence to: Lydia M. Henderson, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, UK BS8 1TD. Fax:44 117 9288274 E-mail:l.m.henderson{at}bristol.ac.uk.
Released online: 15 November 1999
![]() |
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 (O2·2). Suspensions of CHO91 cells exhibit arachidonate-activatable H+ fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:59095916) 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. 351:199216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:15901598), a lowered external pH (pHo) 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 µM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 1020 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 (pHi) was ~6.9, as though pHi was largely determined by endogenous cellular regulation. Arachidonate (20 µM) 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 NH2-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. 19:13191327) and the transfer of protons across an "H-X-X-X-H-X-X-X-H" motif lining a conducting pore.
Key Words: voltage-gated H+ pathway, proton current, gp91-phox, NADPH oxidase, Zn2+
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neutrophils generate superoxide (O2·2) during phagocytosis of opsonized bacteria, immune complexes, and other bodies (
Analysis of genetic lesions in patients with chronic granulomatous disease (CGD)1 has facilitated the identification of the components of the phagocytic NADPH oxidase (phox). There is considerable evidence that one component, gp91-phox, a product of the X-linked CGD gene (
A likely candidate for the H+-selective pathway is the voltage-gated H+ conductance first described in giant molluscan neurons (
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction and Maintenance of CHO Cell Lines
The stable CHO cell line expressing the full-length gp91-phox (CHO91) was constructed and cultured as described previously (
Whole Cell Recordings in CHO Cell Lines
Cells were studied in the whole cell configuration under conditions designed to maximize the amplitude of H+ currents present (see and the seal resistance was in the order of 13 G
at the outset. Cells were bathed in a saline that contained 110 mM TMA methane-sulphonate, 2 mM Ca(OH)2, 2 mM Mg(OH)2, 5 mM glucose, and 100 mM pH buffer. The pH was adjusted to 8.0 or 7.5 with N-[2-hydroxyethyl]-piperazine-N'-[3-propane-sulphonic acid] (EPPS) or 7.0 with HEPES. Other recordings were made from cells bathed in 120 mM NaCl, 10 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 50 mM HEPES, 1 mM NaH2PO4, 5 mM glucose, pH 8.0. Cells were superfused with up to seven different solutions using an in-house superfusion system (
Equilibration of Pipette Filling Solution with Cell Cytoplasm
To produce a transient 1-U change in the pH of cell cytoplasm, it is necessary to inject at least 10 mmol H+/liter (
The time taken for the pipette filling solution to equilibrate with the cell cytoplasm was assessed using a confocal optical scanning microscope (MRC 600; Bio-Rad Laboratories). Because the microscope collects emitted fluorescent light only from within the plane of focus of the objective lens, it performs noninvasive optical sectioning (
Calibration of pHi
Confocal images were collected as above with the pH sensitive fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; 50 µM) in place of Lucifer yellow. Images of patch pipettes filled with 50 µM BCECF dissolved in pH 6.1, 6.5, and 7.0 pipette solutions were used to calibrate the BCECF fluorescence intensity.
Expression and Cellular Localization of Mutated gp91-phox
NH2-terminal mutants of gp91-phox were constructed with three tandem copies of the hemagglutinin (HA) epitope on the COOH terminal of the protein (
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the work reported here, 25-µm-diameter CHO cells were dialyzed against 2-µm-diameter patch pipettes. In view of the difficulty in controlling pHi (see MATERIALS AND METHODS) preliminary experiments were designed to (a) establish the time course of the exchange between pipette and cell, and (b) determine the final level of pHi.
Control of Intracellular pH
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 ( 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 (Figure 1 C). There was no change in cytoplasmic fluorescence intensity if the cell membrane remained intact (not shown).
|
To examine the time course of the change in pHi, we used patch pipettes filled with the pH indicator BCECF buffered with 120 mM pH buffer (Mes). The molecular weight of BCECF (520) is similar to Lucifer yellow, while Mes (213 D) is somewhat smaller and should equilibrate with the cell more rapidly than the dye. The emitted fluorescence intensity of BCECF decreases with decreasing pH and so the cytoplasm should register only a small overall increase in fluorescence if its pH followed that of the pipette solution (pH 6.2). Figure 2A and Figure B, shows that the time course of the fluorescence increase was similar to that for Lucifer yellow, fluorescence reaching a maximum in 1520 min. However, unlike Lucifer yellow, the fluorescence intensity of the cell and the pipette were not equal once the system had reached a steady state (Figure 2 C) and remained different even after 60 min in the whole-cell configuration. The average fluorescence intensity of six cells (after 20 min in the whole cell configuration) was 163 U compared with 91 U for the pipette contents. Assuming that BCECF, like Lucifer yellow, was equally distributed between cell and pipette, and also that there was no interaction between BCECF and the cytoplasm, the inequality of fluorescence must arise from a difference in pH between the pipette solution and the cell contents. From the calibration curve (Figure 2 D), the average value for pHi was near 6.9. As this was significantly different to the pipette solution (buffered to pH 6.2), it seemed that pHi was largely determined by endogenous cellular regulation. Nevertheless, the level of acidification achieved by using pipettes filled with 120 mM pH buffer was sufficient for the purposes of the present experiments. In control cells loaded with BCECF, pHi at rest is estimated to be 7.2 (
|
Whole Cell Currents in CHO and CHO91 Cells
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 (
|
Under identical conditions, CHO91 cells expressing full-length gp91-phox generated large time- and voltage-dependent currents. The outward currents were not recorded immediately upon going whole cell, but increased with time, reaching a maximum after 1025 min. This is in good agreement with the time course of exchange between the cell and the patch pipette shown in Figure 1 and Figure 2 and suggests that the amplitude of the current was not just a property of the transfected cells, but depended on the degree of acidification of the cell cytoplasm. This was confirmed by experiments in which the pipette contents were buffered to pH 7.5 with EPPS buffer (not shown). In this case, there was no change in the whole cell current during a prolonged period of recording.
Small differences in the expression of gp91-phox may explain the observed variation in current amplitude, but there were also differences in time course. Figure 3 C shows a cell in which the current steadily increases throughout each 800 ms command step, while in Figure 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 1.74.6 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 1.57.0 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.
|
|
|
Activation by Sodium Arachidonate
The H+ pathway associated with NADPH oxidase (
As shown in Figure 4 B, sodium arachidonate appeared to shift the voltage dependence of activation towards more negative membrane potentials. The membrane conductance at the end of each command pulse was calculated assuming -55 mV for the equilibrium potential (see later for tail current reversal potential) and the maximum value normalized to 1. The Boltzmann curve (solid line) fitted to the data obtained in 20 µM sodium arachidonate (Figure 4 B, ) has a slope factor of 20 mV with half-activation (V1/2) at -2 mV. The control data (Figure 4 B, ) when normalized to the same maximum value were described by a second Boltzmann curve with the same slope factor and a V1/2 of 21 mV. The maximum conductance in the control was 0.87x that found in the presence of sodium arachidonate. It appears that the effect of arachidonate is both to increase the maximum conductance available and shift the activation curve to more negative values.
The shift of the activation curve to more negative voltages in the presence of arachidonate as shown in Figure 4 B (see also
Inhibition by Zn2+
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 (
Outward Current at Different External pH
Studies on preparations as different as snail neurons and human neutrophils have shown that the voltage dependence of the H+ conductance is shifted to more negative potentials by low pHi and high pHo (
Reversal Potential of the Tail Currents
In Figure 3 and Figure 4, the time-dependent outward currents were rapidly deactivated after each command step, and this was seen as an outward "tail" when the cell was repolarized to -40 mV. To establish whether H+ was the charge-carrying species, tail currents were measured in different external solutions and their reversal potential was determined. For this series of experiments, the pipette solution was buffered to pH 6.5. For Figure 6 A, pHo was set to 8.0 and the cell was depolarized to 0 mV to activate the outward conductance. Repolarization to -100 and -80 mV resulted in a marked inward tail current, but with the potential at -60 mV the current was clearly outward. When repolarized to 40 mV, some deactivation was evident, but there was also a small maintained outward current (as predicted by the activation curve, Figure 4 B). Note that measurements at potentials more positive than approximately -40 mV were precluded because of the voltage activation range of the pathway. The decline in tail current measured over 200 ms was plotted against potential in Figure 6 D ().
When trials were repeated with cells bathed in pH 7.5 saline, inward tail currents were observed not only at -100 and -80 mV, but also at -60 and -40 mV, and so it was clear that the reversal potential had moved in the positive direction (see Figure 6 B). As before, the decline in tail current was measured over 200 ms and plotted against potential in Figure 6 D (). CHO91 cells in pH 7.0 buffered saline generally had a high leakage conductance after 30 min recording and their time-dependent currents were small so that it was necessary to depolarize the cell to 120 mV for significant outward current. Activation was markedly slower in this solution. The decline in tail current amplitude was measured as before and plotted as in Figure 6 D (
). In both pH 7.5 and 7 solutions, it was not possible to generate outward tail currents because of the voltage-activation range of the pathway. A large decrease in the rate of activation with acidification of the external medium has been reported previously (see Figure 5 B3 in
In Figure 6 D, the lines through the data were drawn according to a constant field equation (), scaling factor, 1; pH 7.5 (), scaling factor, 3.5; pH 7.0 (
), scaling factor, 11. The reversal potential in each solution was estimated by simple extrapolation and in pHo8 solution its mean ± SD was -55 ± 6 mV (n = 18). In pHo 7.5 solution, the average reversal potential was -20 ± 3 mV (n = 9) and, in pHo 7 solution, it was 1 ± 5 mV (n = 6). The good correspondence between this data and the 58 mV change/pH unit predicted by the Nernst equation confirms that H+ (or an H+ equivalent) is the probable conducting species. Chloride ions are unlikely to make a contribution because neither the amplitude of the outward current nor the tail current reversal potential was affected by bathing solutions containing different concentrations of chloride (0, 1, 2, 4, and 120 mM; not shown). However the Goldman-Hodgkin-Katz equation predicts larger inward currents as the pH of the bathing solution is made progressively more acid. In practice, because of the shift in the activation curve, the currents were significantly smaller in acid bathing solution and increasing degrees of scaling were necessary to fit the data.
Whole Cell Currents in Mutated gp91-phox
A number of CHO cell lines have been constructed that express mutated forms of gp91-phox (
The whole-cell current traces in Figure 7 A show that in transfected but not induced CHO-N cells, as in CHO91 cells under similar conditions, any voltage-gated current present is concealed within the noise level of the recording. Figure 7 A shows data (typical of six observations) from a single cell bathed in pH 8.0 saline and depolarized by a series of voltage commands (see inset at top of figure). CHO-N cells induced to express the NH2 terminus, generated large time- and voltage-dependent currents (Figure 7 B) in response to depolarizing voltage commands in the range 0 to +120 mV. Just as for CHO91 cells, the outward currents were not recorded immediately upon going whole cell, but increased progressively over a 1025-min period (n = 10). The mean current amplitude at the end of a 400-ms command to +80 mV was 2.3 nA (±0.53 nA; n = 9).
|
Outward currents from CHO-NLeuexpressing cells, like those from CHO-N cells, became activated by depolarizing commands to potentials in the range 0 to +120 mV, but they were of a reduced amplitude and slower rise time (Figure 7 C). In these cells, the mean current amplitude at the end of a 400-ms command to +80 mV was 0.61 nA (±0.4 nA; n = 6).
In CHO-N3Leuexpressing cells, the outward currents had an even lower amplitude (see Figure 7 D, which shows a cell with the largest outward currents) in part because activation was shifted to more positive voltages; in most cases, little or no current was observed at potentials more negative than +80 mV. At this voltage, the mean current amplitude at the end of a 400-ms command was <0.01 nA (n = 6); at +140 mV, the mean current was ~0.3 nA.
The tail currents recorded at -40 mV from both CHO-NLeu and CHO-N3Leu cells, in pH 8.0 bathing solution, were inwardly directed, unlike those recorded from CHO- (Figure 6) and CHO-Nexpressing (Figure 7 B) cells, which were outward. In CHO-N3 cells, the mean reversal potential was 23 mV (±9 mV; n = 3). This apparent shift in tail reversal potential suggests an alteration in the selectivity of the conducting pathway when histidine 115 was replaced by leucine.
From the fluorescence intensity of immunostained cells, we were able to compare levels of expression in different mutated forms of gp91-phox. The cDNA constructs have three tandem repeats of the hemagglutinin epitope attached to their COOH-terminal ends. In Figure 8, confocal images of Cd2+ induced CHO-N (A and B) and CHO-N3Leu (D and E) cells immunostained with antihemaglutinin antibody gave an annular pattern of fluorescence that was not observed in uninduced cells (C and F). This pattern of staining implies that the antigen is not only expressed, but that it is located at or in the plasma membrane and it is similar to that already observed in CHO cells expressing full-length gp91-phox (
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Voltage-gated H+ Conductance
In this paper, we conclude that gp91-phox, the product of the X-linked CGD gene and component of the phagocytic NADPH oxidase, functions as a voltage-dependent H+ conductance. Transfected CHO cells expressing gp91-phox exhibit large time- and voltage-dependent outward currents not present in untransfected or nonexpressing cells (see Figure 3). The currents were observed under conditions that minimized a contribution from the smaller ions normally present in physiological saline. The main cation present both internally and externally was tetramethylammonium ion, while the main anion was methane-sulphonate. The tail current that followed a depolarizing command had a reversal potential that depended on the pH gradient across the cell membrane, changing by ~58 mV for a unit change in pHo. Consequently, the charge carrier for these outward currents is likely to be either H+ or an H+ equivalent.
Voltage-gated H+ conductances have been studied in a wide range of different tissues and animal species that include molluscan neurons, amphibian eggs, human neutrophils, and other phagocytic cells (for reviews, see
Comparison with Human Neutrophils
The voltage-gated H+ conductance described by
Although we would expect the gp91-phoxassociated H+ conductance to have properties broadly similar to those found in vivo in neutrophils, the CGD gene product that we are using is one subunit of the oxidase complex, which normally has four or five protein components. Consequently, even if these other subunits do not directly contribute to the proton pathway, we would expect the environment of the expressed product in CHO cells to be significantly different from that in the natural state. This may account for their activation kinetics, which is the most marked difference between the gp91-phoxassociated currents and the proton currents recorded from neutrophils (
Arachidonic Acid Sensitivity
Figure 4 shows that the voltage dependence of the gp91-phoxassociated H+ conductance could be described by a Boltzmann distribution with a slope factor of 20 mV. The effect of 20 µM sodium arachidonate was to shift the voltage dependence of the conductance by 19 mV to more negative potentials and also to increase (1.2x) the maximum H+ conductance available. Both effects took place without appreciably changing the slope of the voltage relationship. In neutrophils, 50 µM sodium arachidonate has a comparable effect, although here a change in the slope factor from 14.7 to 7.5 mV was reported. The activation curve shifts by 1423 mV and there is an approximately threefold increase in the maximum conductance (
Under normal experimental conditions, the relative positions of the activation curve and the position of the tail current reversal potential (equivalent to the H+ equilibrium potential, EH) means that a maintained inward proton current is impossible to record. The large shift in the voltage dependence of activation produced by arachidonate, which is seen in both neutrophils and transfected CHO cells, means that the pathway may be activated even when the membrane is more negative than the EH (see Figure 4 C).
The effect of arachidonate on the gp91-phox current differs somewhat from its effect in peritoneal macrophages, however. In macrophages, arachidonate not only shifts the activation curve to more negative membrane potentials and increases the maximum conductance, but also accelerates the rate of rise of the outward current (
Zn2+ Sensitivity
The inhibition by Zn2+ of the H+ conductance in neutrophils occurs in the same concentration range as we report here for the gp91-phoxassociated conductance (see Figure 5). In electrophysiological experiments on single cells, almost full inhibition is reported at ~100 µM (
Voltage Dependence of Activation
In Figure 3, the data are fitted by a Boltzmann function with a 20-mV slope factor and half-activation voltage under control conditions (pHo 8, pHi 6.9) of ~21 mV. In human neutrophils,
Voltage-dependent Conductance of Mutated gp91-phox
The 230 NH2-terminal amino acids of gp91-phox when expressed in CHO cells exhibit an arachidonate-activated H+ flux that is significantly reduced when leucine is used to replace the histidine residue in position 115 (cell line CHO-NLeu;
The alteration in properties of the outward currents observed in CHO-N3Leuexpressing cells suggest that the histidine residues on either side of histidine-115 also contribute to H+ permeation. If, as the shift in the voltage dependence of activation suggests, one or the other histidine contributes to the voltage sensitivity of the proton conductance, the large shifts in the activation curve with changes in either pHo or pHi become understandable. An alkaline shift in the pH of the intracellular fluid would have the effect of reducing the charge on any histidine residue to which there is access. The observed positive shift in the activation curve corresponds to the effect of substituting the uncharged leucine for the charged histidine. Yet to be explained is the effect of changes in external pH, which is in the opposite direction.
Function of gp91-phox
Neutrophils provide the first cellular immune response of the body to invading micro-organisms. They are attracted to a site of infection where they engulf antibody-coated bacteria, killing and digesting them. That the generation of superoxide by the NADPH oxidase is a major contributor to the process is evident from the susceptibility to infection demonstrated by CGD patients. In vivo, gp91-phox probably functions as a charge compensator for the electron efflux generated upon production of superoxide. It also prevents a large and rapid fall in pHi caused by the coincident release of H+ internally.
NADPH oxidase activity is stimulated by a number of physiological and nonphysiological stimuli such as phorbol esters, unsaturated fatty acids (such as arachidonic acid), and formyl-Met-Leu-Phe. Thus, the large pH gradients and positive voltages used experimentally here were necessary to activate a significant H+ current only in the absence of arachidonate. After the oxidase is activated in vivo, an outwardly directed electron flux, measured by
Structure of gp91-phox
This is the first voltage-gated H+ conductance to be described at the protein level. The amino acid sequence for gp91-phox is unlike any other protein (
Stationary noise analysis of the voltage-gated H+ current in cultured human muscle suggests that the elementary conductance is <0.1 pS (
Such low conductances follow naturally from the low H+ concentrations on either side of the cell membrane and are consistent with the pathway being either a continuous channel across the cell membrane or a carrier site becoming exposed to first one and then the other membrane surface. In the latter case, it is possible that the mechanism of H+ flux through gp91-phox may involve a cycle of protonation/deprotonation with histidine-115 being exposed alternately to the interior and exterior faces of the cell membrane, as described by
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Mr. A. Laude and Mr. L. Brown for their help with some of the experiments; Mr. Michael Rickard provided computing support.
L.M. Henderson is an Arthritis Research Campaign (ARC) Postdoctoral Research Fellow and the patch-clamp amplifier and equipment was funded by an ARC equipment grant.
Submitted: 2 April 1999
Revised: 20 October 1999
Accepted: 21 October 1999
BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; CGD, chronic granulomatous disease; CHO, Chinese hamster ovary; EPPS, N-[2-hydroxyethyl]-piperazine-N'-[3-propane-sulphonic acid]; TMA, tetraethylammonium
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barish, M.E., Baud, C. 1984. A voltage gated hydrogen ion current in the oocytes membrane of the axolotol, Ambystoma. J. Physiol 352:243-263[Abstract].
Bernheim, L., Krause, R.M., Baroffio, A., Hamann, M., Kaelin, A., Bader, C-R. 1993. A voltage-dependent proton current in cultured human skeletal muscle myotubes. J. Physiol 470:313-333[Abstract].
Byerly, L., Meech, R., Moody, W. 1984. Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymnaea stagnalis. J. Physiol. 351:199-216[Abstract].
Byerly, L., Moody, W.J. 1986. Membrane currents of internally perfused neurons of the snail, Lymnaea stagnalis, at low intracellular pH. J. Physiol 376:477-491[Abstract].
Byerly, L., Suen, Y. 1989. Characterization of proton currents in neurones of the snail, Lymnaea stagnalis. J. Physiol. 413:75-89[Abstract].
Cherny, V.V., Henderson, L.M., DeCoursey, T.E. 1996. Proton and chloride currents in Chinese hamster ovary cells. Biophys. J. 70:A77.
DeCoursey, T.E., Cherny, V.V. 1998. Temperature dependence of voltage-gated H+ currents in human neutrophils, rat aveolar epithelial cells, and mammalian phagocytes. J. Gen. Physiol 112:503-522
DeCoursey, T.E., Cherny, V.V. 1996. Effects of buffer concentration on voltage-gated H+ currents: does diffusion limit the conductance? Biophys. J 71:182-193[Abstract].
DeCoursey, T.E., Cherny, V.V. 1993. Potential, pH and arachidonate gate hydrogen ion currents in human neutrophils. Biophys. J. 65:1590-1598[Abstract].
DeCoursey, T.E., Cherny, V.V. 1994. Voltage-activated hydrogen ion currents. J. Membr. Biol 141:203-223[Medline].
Demaurex, N., Grinstein, S., Jaconi, M., Schlegel, W., Lew, D.L., Krause, K.-H. 1993. Proton currents in human granulocytes: regulation by membrane potential and intracellular pH. J. Physiol 466:323-344.
Eder, C., H-G. Fischer, U., Hadding,, Heinemann, U. 1995. Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor. Pflügers Arch. 430:526-533.
Edwards, S.W. 1994. Biochemistry and Physiology of the Neutrophil. London, UK, Cambridge University Press.
Goldman, D.E. 1943. Potential, impedance and rectification in membranes. J. Gen. Physiol. 27:37-60
Henderson, L.M. 1998. Role of histidine identified by mutagenesis in the NADPH oxidase-associated H+ channel. J. Biol. Chem 273:33216-33223
Henderson, L.M., Banting, G., Chappell, J.B. 1995. The arachidonate-activable, NADPH oxidase-associated H+ channel. Evidence that gp91-phox functions as an essential part of the channel. J. Biol. Chem. 270:5909-5916
Henderson, L.M., Chappell, J.B. 1996. NADPH oxidase of neutrophils Biochim. Biophys. Acta. 1273:87-107[Medline].
Henderson, L.M., Chappell, J.B. 1992. The NADPH-oxidase-associated H+ channel is opened by arachidonate. Biochem. J. 283:171-175[Medline].
Henderson, L.M., Chappell, J.B., Jones, O.T.G. 1987. The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem. J. 246:325-329[Medline].
Henderson, L.M., Chappell, J.B., Jones, O.T.G. 1988a. Superoxide generation by the electrogenic NADPH oxidase of human neutrophils is limited by the movement of a compensating charge. Biochem. J. 255:285-290[Medline].
Henderson, L.M., Chappell, J.B., Jones, O.T.G. 1988b. Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel. Biochem. J. 251:563-567[Medline].
Henderson, L.M., Thomas, S., Banting, G., Chappell, J.B. 1997. The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox. Biochem. J. 325:701-705[Medline].
Hodgkin, A.L., Katz, B. 1949. The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108:37-77.
Kapus, A., Romanek, R., Qu, A.Y., Rotstein, O.D., Grinstein, S. 1993. A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages. J. Gen. Physiol. 102:729-760[Abstract].
Kapus, A., Romanek, R., Grinstein, S. 1994. Arachidonate acid stimulates the plasma membrane H+ conductance of macrophages. J. Biol. Chem. 269:4736-4745
Langton, P.D. 1993. A versatile superfusion system suitable for whole-cell and exercised patch clamp experiments. J. Physiol 467:244P.
Lukas, G.L., Kapus, A., Nanda, A., Romanek, R., Grinstein, S. 1993. Proton conductance of the plasma membrane: properties, regulation, and functional role. Am. J. Physiol. 265:C3-C14
Mahaut-Smith, M.P. 1989. The effect of zinc on calcium and hydrogen ion currents in intact snail neurones. J. Exp. Biol. 145:455-464.
Mathias, R.T., Cohen, I.S., Oliva, C. 1990. Limitations of the whole cell patch clamp technique in the control of intracellular concentrations. Biophys. J. 58:759-770[Abstract].
Meech, R.W., Thomas, R.C. 1980. Effect of measured calcium chloride injection on the membrane potential and internal pH of snail neurons. J. Physiol 298:111-129[Abstract].
Oliva, C., Cohen, I.S., Mathias, R.T. 1988. Calculations of time constants for intracellular diffusion in whole cell patch clamp configuration. Biophys. J. 54:791-799[Abstract].
Pusch, M., Neher, E. 1988. Rates of diffusional exchange between small cells and a measuring patch pipette. Pflügers Arch 411:204-211.
Reaves, B., Banting, G. 1994. Overexpression of TGN38/41 leads to mislocalisation of 7- adaptin. FEBS Lett. 351:448-456[Medline].
Roos, D. 1994. The genetic basis of chronic granulomatous disease. Immunol. Rev. 138:121-157[Medline].
Royer-Pokora, B., Kunkel, L.M., Monaco, A.P., Goff, S.C., Newburger, P.E., Baehner, R.L., Cole, S., Curnutte, J.T., Orkin, S.H. 1986. Cloning the gene for an inherited human disorderchronic granulomatous diseaseon the basis of its chromosomal location. Nature. 322:32-38[Medline].
Schrenzel, J., Serrander, L., Banfi, B., Nusse, O., Fouyouzi, R., Lew, D.P., Demaurex, N., Krause, K.-H. 1998. Electron currents generated by the human phagocyte NADPH oxidase. Nature. 392:734-737[Medline].
Schrenzel, J., Lew, D.P., Krause, K.-H. 1996. Proton currents in human eosinophils. Am. J. Physiol 271:C1861-C1871
Shotton, D., White, N. 1989. Confocal scanning microscope: 3D biological imaging. TIBS (Trends Biochem. Sci.). 14:435-439[Medline].
Starace, D.M., Stefani, E., Bezanilla, F. 1997. Voltage-dependent proton transport by the voltage sensor of the Shaker K+ channel. Neuron. 19:1319-1327[Medline].
Thomas, R.C. 1976. The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J. Physiol. 255:715-735[Abstract].
Thomas, R.C., Meech, R.W. 1982. Hydrogen ion currents and intracellular pH in depolarized voltage-clamped snail neurones. Nature. 299:826-828[Medline].
Wanke, E, Carbone, E., Testa, P.L. 1979. K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane. Biophys. J. 26:319-324[Abstract].