letters to nature

oxidase. However, there could be a coincidental activation of

NADPH-oxidase-unrelated currents, or the activation of currents
Electron currents generated through products of NADPH oxidase (reactive oxygen species).
Omission of NADPH from the pipette solution markedly
by the human phagocyte decreased the size of the currents (peak current density:
2:75 6 0:34 pA=pF; n ¼ 28; P , 0:0001) and there was no detect-
NADPH oxidase able NBT staining under these conditions (Fig. 1b). Cytosolic

Jacques Schrenzel*†, Lena Serrander*, Botond Bánfi*‡,

Oliver Nüße*, Reyhaneh Fouyouzi*, Daniel P. Lew*,
Nicolas Demaurex§ & Karl-Heinz Krause*
NADPH is important for the observed currents. The residual
currents in the absence of pipette NADPH are probably due to
endogenous NADPH, which is present in the cytosol directly after
break-in but which is rapidly consumed and/or lost to the patch
8
* Division of Infectious Diseases, Department of Medicine, University Hospital, pipette during the course of the experiment. Pretreatment of cells
and § Department of Physiology, University Medical Center, 1211 Geneva 4, with the NADPH oxidase inhibitor diphenylene iodonium (DPI;
Switzerland ref. 14) strongly diminished the size of the inward currents (peak
.........................................................................................................................
current density in DPI-pretreated cells: 0:42 6 0:22 pA=pF; n ¼ 5)
Electron transport across biological membranes is a well-known and there was no NBT staining (Fig. 1c). In contrast, when super-
feature of bacteria, mitochondria and chloroplasts, where it oxide dismutase and catalase were included in the bath solution, the
provides motive forces for vectorial transport processes1. In inward currents were preserved (peak current density:
contrast, electron transport is generally not found in the plasma 5:11 6 0:76 pA=pF; n ¼ 10), but no NBT staining was seen (Fig.
membrane of eukaryotic cells, possibly because it would interfere
with electric processes at the plasma membrane. An exception is
provided by the phagocyte NADPH oxidase, which generates
.
superoxide (O 2− ) through electron transfer from cytosolic
a

NADPH to extracellular oxygen2–5. The enzyme is essential for +

host defence, and patients with chronic granulomatous disease,
who lack the functional enzyme, suffer from severe infections6,7. It
has been suggested that electron transfer by the NADPH oxidase
might be electrogenic8. Here we demonstrate, using the whole-cell b
patch-clamp technique, the generation of electron currents by the _
NADPH oxidase in human eosinophil granulocytes. The currents
were absent in granulocytes of sufferers of chronic granulomatous
disease and under conditions of low oxygen. Generation of
electron currents across the plasma membrane of eukaryotic
cells has not been observed previously and might be—indepen- c
dently of the generation of superoxide—a physiologically relevant + DPI
function of the phagocyte NADPH oxidase.
On the basis of the amount of superoxide generated by granulo-
+
.
cytes (,10 nmoles O 2− per min per 106 cells9), the NADPH oxidase
should transport ,108 electrons per second per cell. If these electron
fluxes occur through an electrogenic pathway, they would be d
+ SOD / CAT
expected to produce currents of up to 10–20 pA. Such currents
might indeed play a role in the depolarization observed during
granulocyte activation8,10–12. To identify putative currents mediated +
by NADPH oxidase, we analysed whole-cell currents in eosinophils
under experimental conditions designed to minimize currents
e
through known ion channels and transporters. In a first series of + Zn 2+
experiments, we used unbuffered, nominally Ca2+-free solutions in
the bath and pipette (that is, the free Ca2+ concentration was ,5 mM
as determined by Ca2+-sensitive electrodes) and included 8 mM +
NADPH in the pipette solution. Whole-cell patch-clamp recordings
10 pA

at a holding voltage of 0 mV showed the development of inward pipette

currents in 95% of eosinophils (Fig. 1a; peak current density:
6:26 6 0:42 pA=pF; cell capacitance: 2:49 6 0:05 pF; n ¼ 64). To
determine whether the NADPH oxidase was activated under these
conditions, we performed patch-clamp experiments with nitroblue
tetrazolium (NBT) in the bath solution. NBT is reduced by reactive
oxygen species to dark-blue, insoluble formazan–NBT (this repre-
sents positive NBT staining) and is widely used to detect activation
of NADPH oxidase13. 94% of the patch-clamped cells, but none of
the neighbouring unpatched cells, showed dark NBT precipitates
(Fig. 1a). Thus, the inward currents were associated with the
generation of oxygen radicals and their sign and amplitude was
consistent with the predicted electron fluxes through the NADPH
† Present address: Division of Clinical Microbiology, Mayo Clinic, Rochester, 200 First Street SW,
Minnesota 55905, USA.
‡ Permanent address: Department of Physiology, Semmelweis Medical University, H-1444 Budapest 8,
PO Box 259, Hungary.
1 min NADPH
Figure 1 Inward currents associated with activation of NADPH oxidase in human
eosinophils. Whole-cell recordings from eosinophils15 were made in the presence
of 0.25 mM NBT to assay for activity of NADPH oxidase. After current recording
(left panels), the cells were photographed to show the presence or absence of
NBT staining (right panels). a, Pipette solution contained 8 mM NADPH; 15 out of
16 patched cells showed dark NBT precipitates. b, Pipette solution lacked
NADPH; 0 out of 10 cells were NBT-positive. c, Cells were preincubated for
3 min with 10 mM DPI, an inhibitor of NADPH oxidase; 0 out of 5 cells were NBT-
positive. d, Bath solution contained superoxide dismutase (SOD, 50 units per ml)
and catalase (CAT, 2,000 units per ml); 1 out of 10 cells were NBT-positive. e, Bath
solution contained 10 mM Zn2+ to inhibit H+ currents; 5 out of 5 cells were NBT-
positive. Arrowheads mark the onset of the whole-cell recording (break-in); plus
and minus signs indicate the presence or absence of NADPH (8 mM) in the
pipette solution. Bars under right panels indicate 10 mm.

Nature © Macmillan Publishers Ltd 1998

734 NATURE | VOL 392 | 16 APRIL 1998

1d). Thus, superoxide dismutase and catalase removed the oxidase
products without significantly affecting the currents.
To exclude components of H+ currents, we used 10 mM Zn2+ (a
concentration that efficiently blocks eosinophil H+ currents15). As
shown in Fig. 1e, current amplitude and kinetics were nearly
identical to those seen in the absence of Zn2+ (peak current density:
6:58 6 1:97 pA=pF; n ¼ 6), excluding a relevant contribution of H+
currents to the whole-cell currents seen at 0 mV.
What led to activation of NADPH oxidase under our experi-
mental conditions? As the experiments shown in Fig. 1 were
performed using pipette solution without inclusion of Ca2+ buffers,
the activation might be due to elevations of the cytosolic free [Ca2+]
([Ca2+]c). To investigate the possible role of Ca2+ in activation of
NADPH oxidase in patch-clamped cells, we measured [Ca2+]c using
the fluorescent dye indo-1. As shown in Fig. 2a, when using a
nominally Ca2+-free buffer, [Ca2+]c elevations occurred (the indo-1
ratio was 8:1 6 0:4 before, and increased to 10:1 6 0:3 after, break-
in; n ¼ 28). In contrast, when 10 mM BAPTA buffer was included in
the pipette solution, [Ca2+]c rapidly decreased (Fig. 2a; the indo-1
letters to nature
ratio was 7:5 6 0:8 before, and 3:8 6 0:3 after, break-in; n ¼ 5). To
determine whether the observed [Ca2+]c elevation participated in
the activation of the currents, we included Ca2+ buffers in the
pipette solution. Figure 2b shows a dose-dependent block of current
development by Ca2+ buffers. Thus, [Ca2+]c elevations participated
in the activation of the NADPH oxidase under our experimental
conditions.
We next investigated whether the addition of the chemotactic
peptide f-MLP (N-formyl-methionyl-leucyl-phenylalanine) or the
phorbol ester PMA (phorbol-12-myristate-13-acetate; these are
known stimuli of NADPH oxidase activation) to the bath solution
8
could elicit the currents. We added agonist to cells patch-clamped
with 0.2 mM EGTA in the pipette solution (Fig. 2c). Spontaneous
currents were relatively small under these conditions. f-MLP and PMA,
but not the receptor antagonist boc-MLP (N-tert-butoxycarbonyl-
methionyl-leucyl-phenylalanine), clearly activated the currents.
Both the small spontaneous currents and the agonist-stimulated
currents were blocked by addition of DPI, whereas Zn2+, the H+-
channel inhibitor, did not prevent development of the currents.

a c DMSO DPI a b

Current density (pA/pF)

Control G6PD 10
12 _ 6 NADPH
8 _
Indo-1 ratio

64
10 f-MLP
6 +
10 pA

8 4
BAPTA + 28

6 4
1 min 2 5
0 1 2 3 boc-MLP 5 4 4 4 6
0
Time (min) CGD 1 2

—O
r

2
D
_

Ct
b

6P

D
CG

CG
G
PMA
8
64 +
Current density (pA/pF)

+Zn 2+ c
Positive cells (%)
4 PMA
CGD 2
7
_ Control
98 95
13 (n=253) (100)
10 pA

0 + Carrier
45 62
(213) (21)
0 0.05 0.2 10
Ca 2+ buffer (mM) 1 min O 2 depleted CGD 0 0
_ (211) (31)

Figure 2 Activation of NADPH-oxidase currents by cytosolic [Ca2+], receptor
agonists, and phorbol esters. Whole-cell currents and [Ca2+]c elevations were
measured under different conditions of Ca2+ buffering. a, [Ca2+]c recording of cells
2+
patched using a non-buffered (control) or heavily Ca -buffered (10 mM BAPTA)
pipette solution. Data are shown as a ratio of indo-1 fluorescence emission. b,
Peak whole-cell current densities in cells patched with the following pipette
solutions (from left to right): nominally Ca2+-free; 50 mM indo-1 free acid; 0.2 mM
EGTA; and 10 mM BAPTA. The number of analysed cells is indicated above the
columns. c, Currents induced by the application of soluble stimuli to cells
perfused with pipette solutions containing 0.2 mM EGTA. Conditions were, from
top to bottom: control vehicle (dimethyl sulphoxide, DMSO, 0.1%); chemotactic
peptide (f-MLP, 100 nM); receptor antagonist (boc-MLP, 1 mM); and phorbol ester
(PMA, 5 nM) added in the presence or absence of Zn2+ (10 mM) in the bath
solution. Arrowheads indicate the time of addition of the stimulus (2 min after
break-in) and of the inhibitor DPI.
+ NBT test Current
NADPH
Figure 3 Currents in eosinophils from patients with genetic defects. Whole-cell
currents were activated using pipette solutions lacking Ca2+ buffers and
containing either no NADPH (upper traces in a) or 8 mM NADPH (lower traces
in a). a, Oxidase currents in eosinophils of a patient with G6PD, two patients with
CGD and control eosinophils recorded under low-oxygen conditions. Arrow-
heads mark the onset of the whole-cell recording (break-in). b, Maximal current
densities; conditions as in (a). Ctr, control. The number of analysed cells is
indicated above the columns. c, Percentage of cells showing positive NBT
staining and percentage of cells showing NADPH-oxidase currents in eosino-
phils from healthy volunteers (control, .10 different donors), from a CGD carrier,
and from the two CGD patients.

Nature © Macmillan Publishers Ltd 1998

NATURE | VOL 392 | 16 APRIL 1998 735
letters to nature
Thus, the currents could be activated using pipette solutions with-
out Ca2+ buffers (this situation elicits Ca2+-mediated activation), or
by soluble stimuli added to the bath solution.
We next studied eosinophils from patients with genetic defects
related to superoxide production. Patients with glucose-6-phos-
phate-dehydrogenase deficiency (G6PD) show decreased O 2− pro-
. pipette, the eosinophils of patient 2 displayed small currents
16
duction because of decreased NADPH supply . Without NADPH
in the pipette solution, the eosinophils from G6PD patients showed
a b
DPI
1
2 molecular oxygen). We therefore recorded current from cells in
1 3 3 oxygen-depleted bath solutions17. No current developed without
c pipette solution (Fig. 3a, b).
2 1
10 pA
10 pA
1 min
d f
tr
C C
Control DPI 0 eosinophils, and none of the CGD eosinophils (Fig. 3c). NBT
13 4
—20
13
60 mV
0
—40
(pA)
—80 mV
(pA)
10 pA
e g 60
2s
Control
40
2+
Zn 20
—100
—40
Figure 4 Noise analysis and voltage-dependence of the NADPH oxidase
currents. Whole-cell currents were activated using pipette solutions lacking
Ca2+ buffers and containing 8 mM NADPH. a, Continuous recording of a typical
experiment. The current developed progressively after break-in (arrowhead) and
was blocked by DPI (10 mM). A target voltage protocol (−100 mV to +60 mV; 20 mV
increments) was applied before and after DPI addition. b, Current sweeps (2 s,
filtered at 2.5 kHz, digitized at 10 kHz) taken immediately after break-in (1), during
the peak current (2) and after DPI addition (3) (same cell as in a). c, Current
sweeps as shown in b were analysed for mean current amplitude (white bars) and
mean variance (black bars; n ¼ 5; ref. 23). d, e, Families of currents elicited by
identical voltage protocols (inset; see also a) were obtained before and after
addition of 10 mM DPI, and before and after addition of 10 mM Zn2+. Dotted lines
indicate the zero current level. f, Voltage-independent currents (white bars
represent sustained currents at 0 mV) and voltage-dependent currents (black
bars represent deactivating inward currents seen after a voltage step from 0 mV to
−80 mV) observed: under control conditions; in CGD eosinophils; in oxygen-
depleted cells; after addition of 10 mM DPI; and after addition of 10 mM Zn2+. The
number of analysed cells is indicated under the columns. g, Steady-state current–
voltage relationship (determined at the end of the 7-s voltage pulse) in control
eosinophils or after addition of DPI or Zn2+ (n ¼ 13, 7, and 7, respectively). Residual
currents in cells exposed to DPI and Zn2+ were used for leak subtraction.
736
small currents. However, the currents were fully reconstituted when
NADPH was included in the pipette solutions (Fig. 3a, b). We next
studied two patients with chronic granulomatous disease (CGD).
Without NADPH in the pipette, eosinophils from the two CGD
patients did not show detectable currents. With NADPH in the
(14 6 3% of control), whereas the eosinophils of patient 1 did
not display currents above background levels (Fig. 3a, b).
Electron-transfer currents should depend not only on the pre-
sence of the cytosolic electron donor (that is, NADPH) and a
functional electron-transport chain (that is, NADPH oxidase), but
8
also on the presence of an extracellular electron acceptor (that is,
5 pA
0.5 s oxygen in the bath solution, even when NADPH was included in the
5 pA
2 3 Heterozygous carriers of X-linked CGD do not have a clinically
apparent disease; however, 50% of their eosinophils are unable to
2
generate superoxide (because of X-chromosome inactivation). We
therefore studied eosinophils from a carrier, and compared them
with eosinophils from healthy volunteers and from the two CGD
patients described above. NADPH-oxidase currents were found in
2+
G
D
PI most eosinophils from healthy controls, roughly half of the carrier
2
—O D Zn
7 precipitations were found in most control eosinophils, ,50% of
the carrier eosinophils, and none of the CGD eosinophils (Fig. 3c).
The whole-cell patch-clamp technique provides information not
only about the amplitude and kinetics of currents, but also about
7 underlying unitary events. Indeed, the flux of ions through ion
channels is discontinuous, because of the abrupt opening and
closing of the unitary channels. This leads to characteristic whole-
cell current fluctuations, which can be studied by noise analysis18. In
contrast, electrogenic fluxes through a continuous transport
mechanism are not expected to generate detectable noise. We
therefore analysed the variance of the measured whole-cell currents
at three different times: first, directly after break-in, that is, before
Control relevant current activation; second, at the time of maximal current
amplitude; and third, after current block by DPI (Fig. 4a). Inspec-
DPI tion of the three current traces by eye revealed no visible differences
+100 in noise (Fig. 4b) and no difference in current variance (Fig. 4c).
(mV) These results indicate the absence of detectable channel noise in our
Zn
2+ recordings, and are most compatible with a continuous flow of
electrons through the NADPH oxidase.
We next studied the whole-cell currents during steps to various
target voltages (Fig. 4d–g). The currents remained below the zero-
current level at most voltages (dotted line in Fig. 4d, e). The current
kinetics, however, were complex, because voltage-dependent
activation and deactivation occurred during depolarizing and
hyperpolarizing voltage steps, respectively (Fig. 4d, e). These
voltage-dependent currents were identified as H+ currents on the
basis of the following criteria: first, the kinetics of voltage-dependent
activation and deactivation were similar to those of previously
described H+ currents15,19; second, there was a reversible block by
10 mM Zn2+ (Fig. 4e) (not shown); and third, there was a reversal
potential that followed the H+ equilibrium potential (EH+; not
shown).
The inward currents observed at the beginning of a depolarizing
voltage step and at the end of a hyperpolarizing voltage step
suggested the presence of voltage-independent current compo-
nents. Consistent with the presence of a mixture of voltage-
independent inward currents and voltage-activated outward H+
currents, the steady-state current–voltage relationship (Fig. 4g)
was outwardly rectifying and reversed sign at around EH+. Addition
of DPI completely blocked the inward currents and reduced the
voltage-dependent outward currents (Fig. 4d, g). In contrast,
addition of Zn2+ (Fig. 4e) led to a block of the voltage-dependent
currents and revealed the presence of sustained inward currents,
Nature © Macmillan Publishers Ltd 1998
NATURE | VOL 392 | 16 APRIL 1998

with a linear current–voltage relationship over the whole range of
voltages tested (Fig. 4g). The voltage-independent currents were
identified as NADPH-oxidase currents on the basis of the following
criteria: first, as expected for a unidirectional electron transporter,
the currents were inward over the entire voltage range tested
(−100 mV to +60 mV); second, the currents were blocked by DPI;
third, the currents were resistant to the H+-current inhibitor Zn2+;
and fourth, the replacement of CsCl in the solutions with the
impermeant ions N-methyl-D-glycine (NMDG+) and HEPES− did
not modify the currents (not shown).
A puzzling observation of our study is the low threshold of
voltage activation of the H+ conductance under experimental
conditions that support NADPH-oxidase activity. This can be
seen most clearly during a voltage step from 0 mV to −80 mV in
control cells (Fig. 4d, e). The deactivating tail seen under these
conditions shows that the H+ conductance was activated at 0 mV.
Theoretically, however, the H+ conductance is expected to be
activated at voltages of around +40 mV in the presence of a pH
gradient of 0.5 pH units (ref. 20). DPI shifted the threshold of
voltage activation of the H+ conductance towards the more positive
voltages expected for our experimental conditions, as shown by the
disappearance of the deactivating tail (Fig. 4d, f). No tail currents at
−80 mV (that is, the sign of activated H+ conductance at 0 mV) were
observed with other conditions that precluded NADPH-oxidase
activation (for example, in CGD eosinophils or under conditions of
no oxygen, Fig. 4f). Thus, there is a functional coupling between
NADPH oxidase and the H+ conductance. Further studies will be
necessary to elucidate the mechanism of coupling.
Our study is the first to demonstrate electron currents across the
plasma membrane of a eukaryotic cell. These currents are generated
by the phagocyte NADPH oxidase. The precise functions of the
NADPH oxidase in host defence remain to be defined, however. The
direct product of the NADPH-oxidase reaction, superoxide, is a
relatively unreactive oxygen metabolite and its importance in
microbial killing is unclear4. Our results indicate that the NADPH
oxidase can generate large currents, even against major electrical
gradients (Fig. 4g). This feature is unique compared with other
electrogenic processes and might be—by itself—a biologically
relevant function of the NADPH oxidase. Electron transport is a
biologically relevant function of enzymes in, for example, bacteria,
mitochondria and chloroplasts, where it generates protonmotive
forces necessary for crucial cellular processes such as ATP genera-
tion or transport of protonated molecules1,21. Perhaps electron
currents are involved in analogous vectorial transport in the
phagocyte. M
.........................................................................................................................
Methods
Patients. The G6PD patient was a 36-year-old white male with an erythrocyte
glucose-6-phosphate dehydrogenase activity of 0.25 units per g haemoglobin
(normal range: 3.5–5.5). CGD patient 1 was a 33-year-old white male with X-
linked CGD. CGD patient 2 was a 33-year-old white female with gp47phox-
negative CGD. The carrier was a 35-year-old healthy white female, whose
brother had X-linked CGD22.
Purification. Purification of eosinophils and whole-cell patch-clamp studies15,
noise analysis23, [Ca2+]c measurements24, NBT staining13, and oxygen depletion17
were performed as described.
Solutions. If not indicated otherwise, the following solutions were used: bath
solution: 75 mM CsCl, 50 mM CsOH, 50 mM HEPES/pH 7.1, 10 mM tetraethyl
ammonium chloride (TEACl), 1 mM MgCl2, and 0.1% glucose; pipette
solution: 75 mM CsCl, 50 mM CsOH, 50 mM HEPES/pH 7.6, 10 mM TEACl,
1 mM MgCl2, 1 mM MgATP, and 8 mM NADPH.
Statistics. Data are expressed as mean 6 s:e:m: For statistics, a Wilcoxon sign-
rank test with a level of significance a ¼ 0:05 was used.
Received 23 December 1997; accepted 2 February 1998.
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Physiol. 271, C1861–C1871 (1996).
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rat alveolar epithelial cells is determined by the pH gradient. J. Gen. Physiol. 105, 861–896 (1995).
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Acknowledgements. We thank E. Huggler for technical assistance; L. Bernheim, S. Rawlings, and
M. Rossier for discussions; W. Zimmerli, R. Seger and P. Beris for help with the patients; and N. Mensi
for the G6PD determination. This work was supported by grants from the Swiss National Foundation (to
J.S., K.-H.K., N.D., B.B.), the Janggen Poehn Foundation (J.S.), the Max Cloetta Foundation (N.D.), FEBS
(B.B.), and the Swedish Medical Research Grant (L.S.).
Correspondence and requests for materials should be addressed to K.-H.K. (e-mail: kkrause@cmu.unige.ch).
correction
A late Neanderthal
associated with
Upper Palaeolithic artefacts
Jean-Jacques Hublin, Fred Spoor, Marc Braun,
Frans Zonneveld & Silvana Condemi
Nature 381, 224–226 (1996)
..................................................................................................................................
One of the important morphometric variables assessed in the
comparisons between the bony labyrinths of Neanderthals and
modern humans is the sagittal labyrinthine index. This index
expresses what percentage of the posterior semicircular canal is
situated inferiorly to the plane of the lateral semicircular canal. Its
formula, as given in the legend to Fig. 2 of the above Letter, is
incorrect and should be i=ðs þ i Þ 3 100. M
737