Address correspondence to Dr. Nicolas Demaurex, Department of Physiology, University of Geneva Medical Center, 1 Michel-Servet, CH-1211 Geneva 4, Switzerland. Fax: (41) 22-379-5402; email: Nicolas.Demaurex{at}medecine.unige.ch
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ABSTRACT |
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Key Words: physiology NADPHoxidase zinc phagocyte patch-clamp
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INTRODUCTION |
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The fully activated oxidase complex contains at least two membrane-bound proteins, gp91phox and p22phox, that form the flavocytochrome b558 heterodimer (Wallach and Segal, 1996; phox for phagocyte oxidase). The highly glycosylated gp91phox is a transmembrane protein that comprises putative binding sites for NADPH and flavin cofactors at its COOH terminus (Babior et al., 2002
), as well as two heme groups embedded in the plasma membrane and coordinated noncovalently by four histidine residues within gp91phox third and fifth transmembrane domains (Biberstine-Kinkade et al., 2001
). While gp91phox is the core electron transporting enzyme of the oxidase, p22phox seems to have mainly adaptor function and appears to be required for proper maturation and membrane targeting of the gp91phox subunit (DeLeo et al., 2000
). The activation of the oxidase also requires membrane translocation of four cytosolic proteins: a heterotrimer composed of p67phox, p47phox, and p40phox (Lapouge et al., 2002
), and a small guanosine triphosphatase (GTPase) protein, either Rac1 or Rac2 (Bokoch and Diebold, 2002
). Translocation of the heterotrimer requires the phosphorylation of p47phox and the presence of Rac in its GTP-bound form (for reviews on the oxidase assembly and structure see Babior et al., 2002
; Bokoch and Diebold, 2002
; Vignais, 2002
). Optimal assembly and activation of the oxidase complex is further promoted by arachidonic acid (Doussiere et al., 1996
; Shiose and Sumimoto, 2000
; Foubert et al., 2002
) and by products of phosphatidylinositol 3-kinase (Kanai et al., 2001
; Karathanassis et al., 2002
; Brown et al., 2003
; Stahelin et al., 2003
). The importance of a functional oxidase is underscored by the chronic granulomatous disease (CGD), a disease associated with mutations within one of the phox proteins, predominantly the core gp91phox subunit. CGD patients have impaired phagocytic NADPHoxidase activity and suffer from severe and recurrent infections (for reviews on CGD see Goldblatt and Thrasher, 2000
; Geiszt et al., 2001
).
Sustained activation of the oxidase involves the constant flow of electrons across the plasma membrane, a process that is electrogenic and requires the movement of a compensating charge (Henderson et al., 1987; Schrenzel et al., 1998
). Furthermore, as superoxide anions are being generated by the oxidase, H+ ions are left behind in the cytoplasm as a result of the conversion of NADPH + H+ into NADP+ + 2H+. If uncompensated, the sustained transfer of electrons across the membrane would lead to extreme depolarization and cytoplasmic acidification, conditions that would strongly interfere with the oxidase activity. To account for both charge and pH compensation, the core component of the oxidase, gp91phox, has been proposed very early on to contain a pathway for H+ ions (Henderson et al., 1995
), like mitochondrial cytochromes. Proton efflux could be demonstrated in phagocytes by pH measurements (Kapus et al., 1992
), and was shown to be closely linked with the activation, but not with the redox function, of the oxidase (Nanda et al., 1994
). Direct evidence for voltage-gated proton channels was subsequently provided by patch-clamp recordings in phagocytic cell lines, neutrophils, and eosinophils (DeCoursey and Cherny, 1993
; Demaurex et al., 1993
; Gordienko et al., 1996
; Schrenzel et al., 1996
). Voltage-gated proton channels conduct H+ ions very efficiently down their electrochemical gradient, thereby shunting the voltage and pH gradient across the membrane and are required for sustained activation of the oxidase (Henderson et al., 1988
; DeCoursey et al., 2003
).
Proton channels are activated by depolarization and cytosolic acidification, ensuring a functional coupling to the oxidase. The regulation of the oxidase and of the proton-channel overlap in many respects, as both phagocytic H+ channels and oxidase can be activated by arachidonic acid (Cherny et al., 2001), PMA (DeCoursey et al., 2001
), and by intracellular application of a mixture of ATP, GTP-
-S, and Ca2+ (Schrenzel et al., 1998
; Banfi et al., 1999
). In whole-cell recordings from human eosinophils, oxidase activation by GTP-
-S was associated with H+ currents that activated at lower voltages, and had faster activation kinetics and slower inactivation kinetics, an effect that was not observed in cells from CGD patients lacking the gp91phox or p47phox protein (Banfi et al., 1999
). This interconnection between phagocytic proton channels and oxidase led us (Banfi et al., 1999
) and others (Henderson and Meech, 1999
) to postulate that the H+ pathway is either contained within, or is closely associated with the membrane components of the oxidase. Consistent with the channel nature of gp91phox, heterologous expression of gp91phox and of its recently cloned homologues NOX1 and NOX5 produced voltage-activated proton currents in CHO and HEK-293 cells (Henderson and Meech, 1999
; Banfi et al., 2000
, 2001
). However, these cell lines display endogenous H+ currents, raising the possibility that gp91phox and its homologues might function as channel modulators, rather than forming a channel themselves. Consistent with this hypothesis, reconstitution of a fully functional oxidase in COS-7 cells, which lack endogenous H+ currents, was not associated with measurable proton currents (Morgan et al., 2002
). Because of these conflicting results, it is not clear yet whether gp91phox is a proton channel or a channel modulator, as discussed in detail in a recent series of perspective articles (DeCoursey et al., 2002
; Henderson and Meech, 2002
; Maturana et al., 2002
; Touret and Grinstein, 2002
).
The modulation of proton currents in cells with an active oxidase as well as their up-regulation during gp91phox heterologous expression indicate that, if gp91phox is not a channel, it is closely associated with the channel protein(s). However, purification of cytochrome b558 aggregates from resting phagocytes (Nugent et al., 1989) or copurification of the cytochrome with membrane-bound Rap1 GTPase from activated phagocytes (Quinn et al., 1989
) failed to detect any additional membrane protein other than gp91phox and p22phox. Furthermore, given the importance of proton channels for charge and pH compensation during the respiratory burst, mutations that alter the channel function are expected to strongly affect superoxide production and to cause a CGD phenotype. However, so far no CGD case could be attributed to a protein distinct from the NADPHoxidase complex. Instead, most mutations leading to the CGD phenotype map to the gp91phox core component of the oxidase (Goldblatt and Thrasher, 2000
; Vignais, 2002
). Conversely, all of the known mutations that suppress H+ currents or alter its properties are located within the oxidase complex, preferentially in gp91phox (Banfi et al., 1999
; Henderson and Meech., 1999
; Maturana et al., 2001
). Therefore, at this stage, the simplest and most economical theory is still, in our view, the original postulate that the proton pathway is located within the oxidase complex.
The interaction between proton channels and the NADPHoxidase has so far not been studied under conditions that allows a stringent control of the microenvironment near the plasma membrane. This renders the interpretation difficult, because activation of the oxidase and of its associated channel generates local changes in pH, NADPH, and O2.- which, in turn, modulate the activity of the transport proteins being studied. In this study, we describe measurement of the phagocytic NADPHoxidase-generated electron (e-) current and proton (H+) current in inside-out patches from human eosinophils. To our knowledge, this is the first report that investigates these two functions in a cell-free system. The results obtained with this approach further strengthen the hypothesis that, in human eosinophils, the proton channel and the NADPHoxidase interact either physically or via a membrane-limited mediator.
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MATERIALS AND METHODS |
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For patch-clamp experiments cells were placed on glass coverslip in the bottom of a conical recording chamber. Cells and pipette electrode were visualized using an inverted microscope (Axiowert 10; ZEISS). In sterile filtered solutions high quality seals (>50 G) formed spontaneously after gently attaching the pipette tip to the cell surface followed by a slight, transient suction through the pipette electrode. Stronger and sustained suction was rarely required and resulted in larger patches. Patch excision was achieved by fast, vertical retraction of the pipette tip from the cell surface. In some cases (mainly when strong suction had to be applied and with smaller tip openings) instead of inside-out patch an excised vesicle was formed this way and the pipette tip had to be lifted very briefly (<1 s) into air to establish the right configuration. This intervention, however, often interfered with long-term seal stability. Excised vesicles could be recognized from the anomalous, transient H+ current activation upon depolarization and repolarization. On average >50% of all attempts resulted in high quality inside-out patch. PMA pretreatment, nucleotides in the bathing solution, and NBT in the pipette all lowered the success rate. For applying different bathing conditions two approaches were used. When complete solution change was required the chamber contained 150 µl of recording solution. Solution change was performed within
1.5 min by infusing 1.5 ml of the desired solution from a gravity-driven perfusion system. The excess volume was continuously removed by a suction pump. When bath solutions contained ATP and GTP-
-S the initial working volume was 50 µl and drugs, such as NADPH and DPI, were pipetted directly into the bath followed by thorough mixing (pipetting five times with a 20-µl pipette). Each drug application caused 1% error in the ATP and GTP-
-S concentration, except for the case of 4 mM NADPH application, which caused 5% error. Experiments performed this way were finished within 30 min after the chamber was filled with the bath solution to avoid significant effect of evaporation. Washout of the nucleotides was achieved by perfusing 1 ml nucleotide free solution as described above. Voltage-clamp experiments were performed using Axoptach-1D patch-clamp amplifier (Axon Instruments, Inc.). Pipettes were pulled from borosilicate glass tubing (type GC150F-10; Clark Electromedical) using a P-87 puller (Sutter Instrument Co.). After fire polishing the tip the pipette resistance was 1230 M
when filled with the pH 7.5 solution (composition see below). The resistance of the same pipettes ranged between 36 M
when filled with 150 mM KCl solution, indicating that the pipette tip had a size commonly used in whole-cell experiments (diameter
1 µm). The bath was grounded using an Ag/AgCl pellet. Current data were not routinely corrected for leak unless it was an inherent result of the applied experimental or analytical approach, as described in the results section. Complete absence of H+ or e- current is stated when the measured current is not different from the leak current at a given voltage, as estimated from the holding current measured at a negative, sub-threshold membrane potential (usually -60 mV) in the nominal absence of NADPH. Current was low-pass filtered at 20100 Hz (-3 dB, 8-pole Bessel filter) and digitally sampled at a rate at least 2.5 times the corner frequency of the analogue filter. For data analysis and figure preparation further, software-based (pClamp 8; Axon Instruments, Inc.) noise reduction was performed offline (simulating an 8-pole Bessel filter). Traces in figures are low-pass filtered offline at 5 Hz (-3 dB) unless otherwise specified in the figure legend. Experiments and data storage were performed using software pClamp 6 (Axon Instruments, Inc.), while for data analysis pClamp 8 was used, both running on a PC/AT computer. Online compensation for the full electrode capacitance was not performed, because the stable extent of electrode immersion could not be ensured after a solution change. Continuous current recordings in this paper were neither intended nor suited to perform tail-current analysis, therefore changes in tail-current kinetics are not interpreted, although in most cases they displayed the expected changes, based on earlier reports (see DISCUSSION for details).
Solutions
The cell storage media was a 2:1 mixture of Medium 199 with L-glutamine and L-amino acids containing 25 mM HEPES (GIBCO BRL) and L-glutamine free RPMI 1640 Medium (GIBCO BRL) supplemented with 5 mM Na2EDTA and 2% fetal calf serum (BioConcept). The recording solutions contained (mM): CsCl 1, tetraethylammonium chloride 1, MgCl2 2, EGTA 1, N-methyl-D-glucamine base 101 and either 200 MES acid for pH 6.1 or 200 HEPES acid for pH 7.5. To establish pH 7.0 solution pH 7.5 and pH 6.1 solutions were mixed at a volume ratio of 3:2 (7.5 versus 6.1, respectively). Free [Zn2+] was calculated using MaxChelator software (version 2.10, http://www.stanford.edu/~cpatton/maxc.html, written by Chris Patton) and buffered with 3 mM citrate in the µM range or by the 1 mM EGTA in the nM range. Zinc effect was tested using citrate containing pH 7.5 solution, as HEPES does not bind Zn2+ significantly (Cherny and DeCoursey, 1999). The pH of citrate containing solution with 45 µM free Zn2+ was 7.43. Osmolality of the above recording solutions was in the range of 315322 mosmol/kg, as measured with a freezing point osmometer. The osmotic and volume changes induced by bath application of different compounds were not corrected for. Li4GTP-
-S was dissolved in water at 10 mM. DPI was first dissolved in DMSO to give a 10-mM stock, which was then further diluted in pH 7.5 solution to 200 µM. PMA was dissolved in DMSO at 1 mM. MgATP and Na4NADPH was dissolved in pH 7.5 solution to give a stock solution of 30 and 80 mM, respectively. ZnCl2 was dissolved at 0.5 M in water containing 40 mM HCl. Na2EDTA was dissolved in water at 250 mM. All chemicals were obtained from Sigma-Aldrich unless otherwise specified. All manipulations were performed on room temperature (2325°C), unless otherwise stated.
Conventions
The sign of the originally recorded inside-out patch current signal is inverted in figures; thus, positive current value indicates outward current to comply with classical whole-cell experiments. Intracellular pH (pHi) is the pH of the bath solution facing the cytoplasmic side of the excised patch membrane, while the pH of the pipette solution corresponds to pHo (extracellular pH).
Data Analysis
The number of cells (n) indicates the cumulative number of measurements performed on at least two independent cell preparations. Data are presented as mean ± SD. For statistical analysis paired or unpaired Student's t test and ANOVA for repeated measures was applied using Statistica software (version 4.5; Statsoft, Inc.). For ANOVA post hoc comparisons the Tukey honest significant difference test was applied. A value of P < 0.05 was considered statistically significant. Linear regression and correlation coefficients were calculated using the built-in algorithm of Origin software (version 6.0; Microcal Software, Inc.). Exponential fit of current activation and its extrapolation were performed with the built-in algorithm of pClamp 8 software. For all fits and regressions the sum of squared error minimization method was applied.
Online Supplemental Material
Supplemental figures available online demonstrate the proton selectivity (Fig. S1) and DPI sensitivity (Fig. S4) of depolarization-activated inward current. Further figures depict the run-down of electron current in ATP- and GTP-free solutions (Fig. S2) and the correlation between inward and outward H+ currents and between e- and outward H+ currents (Fig. S3). These measurements were performed in patches excised from PMA-treated eosinophils. Online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200308891/DC1.
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RESULTS |
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The reversibility of the Zn2+ block could be tested directly in cell patches sequentially exposed to different Zn2+ concentrations. Shifting from 100 nM to
3 µM Zn2+ reduced the amplitude of the steady-state H+ current by 69 ± 6.5% (at 80 mV, n = 4, P < 0.0005), whereas subsequent application of
45 µM Zn2+ eliminated any depolarization-activated H+ current within 3 min at 80 mV. These effects were completely reversible.
Proton Current in Excised Patches from PMA-stimulated Eosinophils
PMA is a well-known activator of the phagocytic NADPHoxidase and of its related proton channel (DeCoursey et al., 2000, 2001
). When patches were excised from eosinophils pretreated with supramaximal stimulatory doses of PMA (200400 nM for 510 min), membrane depolarization also activated an outward current in the vast majority of patches (n > 50). The kinetic of activation was, however, much faster than in nontreated patches as steady-state was usually reached within 20 s (Fig. 4
C). This finding is in qualitative agreement with earlier reports that compared the characteristics of whole-cell H+ currents in activated and nonactivated phagocytes (Banfi et al., 1999
; DeCoursey et al., 2000
, 2001
). Unlike whole-cell currents, however, H+ currents in inside-out patches were subject to progressive run-down, which started within 3 min after patch excision and usually stopped within 10 min (Fig. 4 A). Unexpectedly, stepwise reductions in current amplitude were regularly observed during run-down (14 of 16 cells, Fig. 4 A, inset). The run-down could be strongly hampered or completely blocked by bath application of 35 mM ATP and 2025 µM GTP-
-S, while washing out these compounds initiated run-down within <1 min (Fig. 4 B). The run-down never resulted in the complete loss of outward H+ current, but instead reverted its activation kinetic and long-term stability to those observed in nonactivated cells (Fig. 4 C).
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DISCUSSION |
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Proton Currents from Nonstimulated Eosinophils and the Effect of Internal Zn2+
In most patches excised from nonstimulated eosinophils, voltage-activated H+ currents could be measured and were the predominant currents observed. The current amplitude and its long-term stability were sufficiently large to perform a functional and pharmacological characterization of the underlying channel, under conditions that allow a stringent control of the pH on both sides of the membrane. The properties of the H+ currents recorded in excised patches were consistent with earlier studies on phagocytic H+ currents performed at the whole-cell level: voltage and pH dependency, exceptionally high selectivity for H+, and direct activation by micromolar arachidonic acid (unpublished data; Kapus et al., 1994; Gordienko et al., 1996
; Schrenzel et al., 1996
, for review on the characteristics of phagocytic and other H+ currents see DeCoursey, 2003
).
The ability to apply agents from the internal side of the membrane allowed us to study the mode of action of Zn2+, which inhibits H+ currents in all cell types at submillimolar concentrations when applied from the external side (DeCoursey, 2003). In nonactivated eosinophils, a near complete block is achieved with a few 10 µM free extracellular Zn2+ (Gordienko et al., 1996
; Schrenzel et al., 1996
; Banfi et al., 1999
), but it is unclear whether the Zn2+ binding sites are located on the extracellular side of the channel or even on the channel itself. In one study, the effect of extra- and intracellular Zn2+ were compared in rat alveolar epithelial cells. Only a moderate inhibition was observed at high intracellular Zn2+ concentrations (
170 µM), values much higher than those required for extracellular block at the same pH of 6.5 (Cherny and DeCoursey, 1999
). The authors concluded that Zn2+ probably has a qualitatively and quantitatively different effect after external or internal application. This appears not to be the case in eosinophils, as a near complete block could be observed at an intracellular free Zn2+ concentration of 45 µM, while 3 µM internal Zn2+ caused more than half maximal inhibition of the steady-state H+ current at 80 mV and pHi 7.5. These data are comparable to that of Schrenzel et al. (1996)
, who tested the effect of external Zn2+ in human eosinophils and reported a half-inhibitory concentration of 4 µM at pHo 7.5 (Schrenzel et al., 1996
). Whether the apparent difference in Zn2+ action between epithelial cell and eosinophil is of methodological or biological origin is yet to be elucidated. Nonetheless, our data suggest that, in nonstimulated human eosinophils, the blocking action of Zn2+ is largely independent of its site of application with the notable exception that, contrary to extracellular application (Cherny et al., 2001
), depolarization promotes the block by internal Zn2+. Therefore, Zn2+ either binds to the same site(s) accessible from both sides of the membrane or to distinct intra- and extracellular sites that are still located within the membrane electrical field. We did not test the influence of pHi on the inhibitory potential of Zn2+, but as most sites that bind Zn2+ do bind proton similarly well, a pHi decrease would probably diminish Zn2+ inhibition as observed with extracellular Zn2+ (DeCoursey, 2003
).
Characteristics and Run-down of Proton Currents in Cells Stimulated with PMA
Earlier whole-cell patch-clamp measurements in phagocytes expressing a functional oxidase consistently show that proton currents have larger amplitude, lower threshold, faster activation, and slower deactivation kinetics when cells are stimulated with GTP--S or PMA to activate the oxidase (Banfi et al., 1999
; DeCoursey et al., 2000
, 2001
). The approximately -40-mV shift in the threshold potential (Banfi et al., 1999
; DeCoursey et al., 2001
) allows the influx of H+ through the channel under appropriate pH conditions (Banfi et al., 1999
), a unique condition among proton channels. Our results from inside-out patches confirm and extend these earlier reports. Large, rapidly activating inward H+ currents could be induced by depolarizing voltage steps in patches excised from cells stimulated with PMA (Fig. 5). In the absence of NADPH the inward and outward H+ currents were of similar amplitude when measured at an identical H+ driving force, and were both associated with excess current fluctuations (Fig. 5). Interestingly, this phenotype ran down within minutes in patches excised from PMA-treated cells, although run-down could be hampered by adding ATP and GPT-
-S to the cytosolic side of the patch. The run-down resulted in a partial loss of outward current amplitude, slowing of the activation kinetic, and complete loss of inward H+ currents. In many cases (e.g., Fig. 4 B) only a single kinetic component was observed when outward H+ currents were elicited at the beginning of the recording and after run-down had stopped. This suggests that the run-down reflects the transition between two functional state of the same channel, as was proposed by others for PMA stimulation (DeCoursey et al., 2001
). Thus, contrary to our earlier proposal based on recordings of eosinophils from CGD patients (Banfi et al., 1999
), normal eosinophils appear to express only one type of H+ channels.
Unexpectedly, the run-down often occurred in an abrupt, stepwise manner. The amplitude of the current steps far exceeded the current size that can be carried by a single H+ channel based on current fluctuation analysis or theoretical considerations (see below), suggesting that these abrupt transitions do not reflect the closing of single proton channels. One possibility is that the stepwise current changes reflect the pinch-off of membrane vesicles from the patch. However, this possibility appears unlikely as vesicle formation and detachment are energy-requiring processes, while the stepwise run-down often began to occur minutes after excising the patch and was observed preferentially in the absence of ATP and GTP. Another possible explanation is that proton channels are clustered in the plasma membrane and display state transitions in a concerted manner, as was shown for many other ion channels (Schindler et al., 1984; Hymel et al., 1988
; Geletyuk and Kazachenko, 1989
; Schreibmayer et al., 1989
; Marx et al., 1998
; Bukauskas et al., 2000
; Wang et al., 2000
; Tanemoto et al., 2002
). Cooperation between H+ channels or channel subunits may also explain the delay and sigmoidicity in the channel activation kinetic that is observed in phagocytes and in alveolar epithelial cells (DeCoursey, 2003
).
The run-down phenomenon provided new evidence linking electron transport to proton channel activity. Both electron and proton currents ran-down with similar kinetics (Fig. S2), and stepwise changes were also observed during electron current fade-out when NBT was present in the pipette (Fig. 6), consistent with clustering of both entities. In addition, the run-down of both proton and electron currents could be partially, and sometimes fully, inhibited by the simultaneous presence of internal ATP and GTP--S. These nucleotides are required to maintain the oxidase activity in cell free systems (Ligeti et al., 1988
; Doussiere and Vignais, 1992
) or in streptolysin-O permeabilized phagocytes (Brown et al., 2003
). High concentrations of ATP may be required to maintain the phosphorylation pattern of the oxidase subunits (Babior et al., 2002
) and/or of membrane phospholipids such as phosphoinositides (Kanai et al., 2001
; Karathanassis et al., 2002
; Brown et al., 2003
; Stahelin et al., 2003
). ATP and GTP, on the other hand, are required to maintain the activity of several ion channels in excised patches. The observation that ATP and GTP-
-S are required to maintain the activity of both electron and proton current in excised patches indicates that the these nucleotides modulate the underlying transport proteins in a similar way. Because GTP-
-S has been shown to bind directly to membrane associated small GTP-ases within the activated phagocytic oxidase complex (Bokoch and Diebold, 2002
), it is tempting to speculate that the proton channel is also part of the oxidase complex. Alternatively, the channel and oxidase might be separate entities sharing a very similar regulation. This is not unexpected, as prolonged function of the oxidase relies on H+ channel activity (Kapus et al., 1993
; Banfi et al., 1999
; DeCoursey et al., 2003
). Further experiments are required to understand the inactivation process underlying the run-down phenomenon and to determine the site of actions of the nucleotides.
Low-frequency Noise Associated with Proton Current
The depolarization activated H+ currents are thought to be carried by voltage-gated proton channels. One of the fundamental arguments that the current is flowing through channels rather than carriers is the presence of excess fluctuation during current activation (DeCoursey, 2003). These fluctuations are thought to denote abrupt transitions between conducting and nonconducting states of the channel, called gating events (Hille, 1992
). Based on statistical considerations, the average conductance of the underlying single channel can be calculated from the measured average macroscopic H+ current amplitude, its variance, and estimated open probability (Hille, 1992
). In patches excised from nonstimulated eosinophils a characteristic increase in current noise could be observed in the low-frequency bandwidth (<10 Hz) during outward H+ current activation (Fig. 1), consistent with a recent study (Cherny et al., 2003
). Our observation that steady-state inward H+ current from activated eosinophils also display similar fluctuation at 0 mV, where no other significant noise source is present (Fig. 5), leaves hardly any doubt that these fluctuations are produced by state transitions of H+ channels. Surprisingly, the estimated single H+ channel conductance derived from these fluctuations (
30140 fS, Cherny et al., 2003
) is at least an order of magnitude larger than predicted if H+ diffusion in the bulk solution was rate limiting. Several hypotheses were proposed to resolve this apparent contradiction, the discussion of which exceeds the scope of this paper (for review see DeCoursey, 2003
). A simpler but as yet not considered explanation might be that H+ channels are in fact clustered, as mentioned above, and might function (gate) in concert.
Relationship between Proton and Electron Currents
There is an ongoing debate about the channel nature of the core subunit of the phagocytic NADPHoxidase, gp91phox, which has been proposed to possess a built-in H+ pathway (Henderson et al., 1995; Banfi et al., 1999
). Unfortunately, this study cannot provide a definite answer as to the identity of the proton channel molecule, but using the inside-out patch approach we could challenge two earlier results obtained at the whole-cell level. In the first series of experiments we tested whether H+ and e- current amplitudes are correlated. No correlation was observed in whole-cell perforated patch measurements between the amplitudes of e- current and of the maximal outward H+ conductance in cells stimulated with PMA, arguing against the H+ channel nature of the oxidase (DeCoursey et al., 2000
). Because outward H+ currents are observed regardless of the activation state of the oxidase, we initially investigated the relationship between e- and inward H+ currents. Like e- currents, the presence of inward H+ current depend on the assembly of oxidase complex, which shifts the voltage threshold of H+ channel activation below EH (Banfi et al., 1999
). We could observe a good correlation between inward H+ and e- currents at saturating concentrations of the substrate, NADPH, or when the reaction product, O2.- was rapidly removed from the extracellular side by a scavenger. Under these conditions, the driving force for e- across the patch of membrane (redox potential difference calculated from the NADPH:NADP+ and O2:O2.- ratios at a given pH) is expected to remain relatively constant. In contrast, at lower [NADPH] and without an external O2.- scavenger the steady-state O2:O2.- ratio at the extracellular membrane surface might strongly depend on the number of active oxidases in the patch, as the pipette tip is a limited diffusion space. This might account for the lack of correlation observed with 0.8 mM NADPH (Fig. 7 B). Similarly, the lack of correlation reported in whole-cell studies might reflect cell to cell variations in the metabolic state, as the intracellular concentration of NADPH cannot be controlled in perforated patch measurements (DeCoursey et al., 2000
). The correlation that we observed in excised patches did not depend on the direction of the H+ flux, as a linear correlation was also observed between the amplitude of e- currents and the amplitude of the outward H+ currents measured in the same patch.
The correlation indicates that the two functions are coupled, but does not ensure the molecular connection between the oxidase and channel proteins. Homogenous distribution of the two molecules can also yield such a correlation if the ratio of expression does not vary too much among patches. However, it was suggested that at least two oxidase complexes have to cooperate in close contact for high-capacity e- transport (Vignais, 2002), and NADPHoxidase complexes tend to distribute in aggregates rather than evenly in phagocytes (Nugent et al., 1989
; Wientjes et al., 1997
). To account for the correlation between the two currents in excised patches, H+ channels must therefore follow a similar distribution pattern.
In the second series of experiments we revisited the evidence indicating that the oxidase blocker DPI does not significantly affect H+ currents. DPI failed to influence the activation kinetic and the amplitude of outward H+ current in whole-cell perforated patch studies, and only reverted the slowing of the tail current induced by PMA (DeCoursey et al., 2000, 2001
). The effect of DPI on the inward H+ current was not reported in these studies, and no convincing explanation could be provided for the DPI effect on tail currents. Because tail currents through proton channels denote inward H+ transport, we tested whether DPI affects the amplitude of steady-state inward proton currents in PMA-pretreated eosinophils. The inside-out patch approach allowed us to analyze systematically the effect of DPI and of NADPH by comparing the H+ current amplitudes in the presence and complete absence of these compounds. Such a protocol could not be applied using the whole-cell technique, and allowed us to separate the direct effects of DPI on H+ currents from its indirect effects caused by the inhibition of oxidase function.
These experiments indicate that only the inward, but not the outward H+ transport is significantly affected by the simultaneous presence of DPI and NADPH on the cytosolic side of the patch membrane (Fig. 8). These observations are consistent with whole-cell results showing that DPI inhibits inward tail currents, but not outward proton transport through the H+ channel. However, the interpretation becomes different, as under our conditions the local pH, [O2.-] and electrical field changes generated by the oxidase are essentially absent when DPI is added before activation of the oxidase by NADPH. Therefore, the inhibition of inward H+ flux observed under these conditions reflects the binding of NADPH and/or DPI. The requirement for reducing conditions for DPI to block the oxidase was observed in a cell-free system (Doussiere and Vignais, 1992) and may explain why a consistent DPI effect can only be observed in the presence of NADPH. A strong inhibition of inward H+ current by DPI could be occasionally observed in the nominal absence of NADPH (Fig. S4), although the average effect was not significant. Together, these data indicate that DPI exerts either an unidirectional or a voltage-dependent block on the PMA-activated state of the proton channel. DPI appears to bind preferentially to the outer heme component of the oxidase (Doussiere et al., 1999
), which becomes released by a coordinating histidine during oxidase activation (Doussiere et al., 1996
). We suggested earlier that this histidine residue mediates inward H+ flux, as the histidine reagent diethyl pirocarbonate blocked inward H+ current activation and speeded up the tail current kinetic. Unlike DPI, however, diethyl pirocarbonate also reduced the amplitude and the activation kinetic of the outward current, suggesting that it reverted the shift in H+ channel voltage dependence induced by oxidase activation. Although we cannot formally rule out the possibility that DPI and/or NADPH also shift back the H+ channel activation threshold to more positive potentials, this possibility appears unlikely as DPI appears to specifically alter inward H+ transport, both in whole-cell and excised patches. Our observations that agents known to bind to gp91phox and to alter the oxidase function also alter proton transport in excised patches containing a functional oxidase further suggest that the proton channel and the oxidase complex in normal phagocyte are closely connected.
In summary, human eosinophils express mainly one type of voltage-dependent proton channel whose function is linked to the NADPHoxidase. Further experiments are required to ultimately prove whether or not the proton channel is contained within the NADPHoxidase complex, but the linear correlation between e- and H+ current amplitude, their similar rundown kinetics, and the effect of oxidase specific agents on H+ currents suggests that the proton channel is either part of the oxidase complex or linked by a membrane-limited component. The ability to reliably measure electron currents in inside-out patches from human eosinophils allows us to directly address yet unresolved issues on the regulation of the phagocytic NADPHoxidase. The inside-out patch technique may be particularly advantageous to study the highly regulated, but as yet mysterious processes involved in the down-regulation and inactivation of the oxidase.
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FOOTNOTES |
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Abbreviations used in this paper: CGD, chronic granulomatous disease; DPI, diphenylene iodonium chloride; NADPH, nicotinamide adenine dinucleotide phosphate; NBT, p-nitro-blue tetrazolium chloride; phox, phagocyte oxidase.
1 The empirical equations that can be applied to estimate the H+ current threshold potential (Ethreshold) under different EH (Erev) conditions in resting and PMA treated eosinophils are Ethreshold = 0.79 Erev + 23 mV and Ethreshold = 0.63 Erev - 22 mV, respectively (DeCoursey, 2003
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ACKNOWLEDGMENTS |
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This work was supported by grant number 31-068317.02 from the Swiss National Science Foundation and by the National Science Foundation of Hungary (OTKA, TS 040865).
Olaf S. Andersen served as editor.
Submitted: 25 June 2003
Accepted: 13 October 2003
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