Escherichia coli oscillations in mammalian cells–the pore is on its own

The FASEB Journal • FJ Express Summary
Why Escherichia coli ␣-hemolysin induces calcium
oscillations in mammalian cells–the pore is
on its own
¨ nver,* Florian Dreyer,*
Andreas Koschinski,*,1 Holger Repp,*,1,2 Baris U
†
Dierk Brockmeier,* Angela Valeva, Sucharit Bhakdi,† and Iwan Walev†,2
*Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University, Giessen, Germany; and
†
Institute of Medical Microbiology and Hygiene, Johannes-Gutenberg-University, Mainz, Germany
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4561fje
SPECIFIC AIMS
The hemolysin of Escherichia coli (HlyA) is prototypic of
a large family of pore-forming toxins that are produced
by many medically important gram-negative pathogens.
It was reported that HlyA deregulates membrane Ca2⫹
channels, thus inducing periodic low-frequency Ca2⫹
oscillations that trigger transcriptional processes in
mammalian cells. Our aim in this study was to delineate
the mechanism underlying the Ca2⫹ oscillations that
are induced by HlyA.
PRINCIPAL FINDINGS
1. HlyA induces nonperiodic Ca2ⴙoscillations
in renal cells
Within ⬃30 s after application of HlyA (5 ng/ml) to
human kidney tubule epithelial (IHKE) cells, intracellular Ca2⫹ concentration ([Ca2⫹]i) increased and exhibited oscillations during further monitoring. It was
previously reported that HlyA provoked periodic Ca2⫹
oscillations in primary rat proximal tubule (RPT) cells,
with a constant periodicity of Ca2⫹ peaks of 12 min.
However, we found no constant periodicities but instead found large variations of oscillation kinetics and
peak-to-peak spans. Examples are shown in Fig. 1A–D
for Ca2⫹ tracings and in Fig. 1E for false-color images of
[Ca2⫹]i. No Ca2⫹ oscillations were induced by the
nonhemolytic HlyA mutant S177C/K564R/K690R,
which is fully bindable to the plasma membrane but
unable to form membrane pores (Fig. 1G). Statistical
analysis of time spans between directly neighboring
Ca2⫹ peaks in Fig. 1A-D and 48 other Ca2⫹ tracings
yielded a most frequent peak-to-peak span (not to be
confused with peak periodicity) of 70 s, whereas spans
above 10 min were very rare. Peak-to-peak span distribution was dependent on HlyA concentration, whereupon higher concentrations led to shorter time spans.
0892-6638/06/0020-0973 © FASEB
2. A periodicity of Ca2ⴙ peaks is pretended
by a power spectrum analysis
We subjected the Ca2⫹ data sets to a power spectrum
analysis, which was previously used to identify HlyAinduced periodic Ca2⫹ oscillations of low frequency.
Despite marked heterogeneity of the original Ca2⫹
tracings and rare occurrence of peak-to-peak spans
above 10 min, this analysis yielded a periodicity of Ca2⫹
peaks with a dominant wavelength of 12.8 min, which
was in remarkable agreement with the previously reported value of 12.0 min. Surprisingly, in Ca2⫹ tracings
monitored in the absence of HlyA and devoid of Ca2⫹
elevations, a periodicity of ⬃12 min was again computed. Furthermore, analysis of a signal consisting only
of white noise superimposed to a discretely drifting
baseline yielded the same periodicity. In conclusion,
power spectrum analysis appeared as an inadequate
mathematical model for a proper interpretation of the
Ca2⫹ data sets.
3. Nifedipine does not inhibit Ca2ⴙ oscillations
When voltage-gated L-type Ca2⫹ channels in IHKE cells
were blocked by nifedipine, HlyA still led to Ca2⫹
oscillations (Fig. 1F). This contrasted with previous data
of RPT cells where nifedipine abolished the Ca2⫹
response to HlyA. A striking phenomenon that we
observed during the Ca2⫹ measurements can account
for this difference. Nifedipine-treated cells that were in
the microscopic field of view for several minutes during
Ca2⫹ imaging, i.e., irradiated by ultraviolet (UV) light,
1
These authors contributed equally to this work.
Correspondence: Frankfurter Str. 107, Rudolf-BuchheimInstitute of Pharmacology, Justus-Liebig-University, 35392
Giessen, Germany. Email: Holger.Repp@pharma.med.unigiessen.de; and Obere Zahlbacher Str. 67, Institute of Medical
Microbiology and Hygiene, Johannes-Gutenberg-University,
55101 Mainz, Germany. Email: walev@mail.uni-mainz.de
doi: 10.1096/fj.05-4561fje
2
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Figure 1. Ca2⫹ oscillations induced by HlyA in
IHKE cells loaded with the fluorescent Ca2⫹
indicator Fura-2/acetoxymethyl ester. A–D)
Cells were exposed to HlyA (5 ng/ml) 3 min
after start of recording. Traces represent examples of time courses of [Ca2⫹]i of 4 single cells.
E) Single frames from an image series of
[Ca2⫹]i. Frames 1– 4 correspond to numbers
1– 4 above trace in A. Traces in A and B were
recorded from cells that are marked with A and
B. F) Superposition of Ca2⫹ responses of 49
IHKE cells in the presence of nifedipine (100
␮M). Application of HlyA (5 ng/ml) is indicated by arrow. Gap represents time when field
of view was changed to an area that had not
been UV irradiated before. G) Ca2⫹ tracing of a
single IHKE cell on consecutive exposure to a
nonhemolytic HlyA mutant (⫽nh HlyA, 25 ng/
ml) and wild-type HlyA (2.5 ng/ml).
did indeed not show Ca2⫹ oscillations after application
of HlyA, whereas Ca2⫹ oscillations were observed in
cells that had not “seen” UV light (Fig. 1F). Thus, the
previously reported suppressive effect of nifedipine on
HlyA-induced Ca2⫹ oscillations was uncovered as experimental artifact. Additionally, HlyA still led to Ca2⫹
oscillations in HEK293 cells when a combination of
blockers of voltage-gated and receptor-operated Ca2⫹
channels was present, including nifedipine (100 ␮M),
Cd2⫹ (2 mM), and SK&F 96365 (25 ␮M).
4. Ca2ⴙ oscillations and pore formation cease rapidly
on removal of HlyA
The effect of HlyA on [Ca2⫹]i is essentially dependent
on the presence of toxin in extracellular medium.
Instantaneously after the start of a washout in HlyAtreated IHKE cells, [Ca2⫹]i decreased and reached
initial concentration after ⬃3 min. Reapplication of
HlyA again led to Ca2⫹ oscillations, which disappeared
after the restart of washout. After the start of washout,
disappearance of Ca2⫹ oscillations and HlyA pores
followed the same time course. This synchronism was
discovered by subjecting single IHKE cells exposed to
HlyA to simultaneous measurement of [Ca2⫹]i and
pore formation, using at the same time Ca2⫹ imaging
and electrophysiological patch-clamp recording. A few
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Vol. 20
May 2006
seconds after the start of washout the number of open
pores decreased and went to zero within ⬃4 min. A
second HlyA application again led to pore formation.
The short life span of HlyA pores was confirmed by
propidium iodide influx measurements. Cells became
permeable to dye during incubation with HlyA, but
membranes returned to the impermeable state shortly
after toxin removal.
5. Ca2ⴙ elevations result from Ca2ⴙ influx through
HlyA pores
By simultaneous monitoring of pore formation and
[Ca2⫹]i in IHKE cells exposed to HlyA, we detected an
increase in [Ca2⫹]i directly after pore opening and a
good temporal correlation between formation and closure of HlyA pores and Ca2⫹ elevations. We hypothesize
that the decrease after an elevation reflects the restoration of cellular Ca2⫹ homeostasis by physiological
Ca2⫹ redistribution after HlyA pore closure (Fig. 2).
CONCLUSIONS AND SIGNIFICANCE
The results of this study lead us to a new explanation
for Ca2⫹ oscillations that occur in mammalian cells
treated with HlyA. Contrary to a previous report, we
showed that the ability of HlyA to induce Ca2⫹ oscilla-
The FASEB Journal
KOSCHINSKI ET AL.
Figure 2. Schematic overview. Ca2⫹ oscillations
induced by HlyA occur only in the presence of
the toxin and are not blocked by nifedipine.
After washout of HlyA, Ca2⫹ oscillations cease
rapidly. Readdition of the toxin again leads to
Ca2⫹ oscillations. We assume that the decrease
after a Ca2⫹ elevation is managed by plasma
membrane and sarco/endoplasmic reticulum
Ca2⫹ pumps (illustrated by circles with blades).
tions does not rely on endogenous L-type Ca2⫹ channels. Furthermore, we found that the effect of HlyA on
[Ca2⫹]i is not dependent on the activity of any voltagegated or receptor-operated membrane Ca2⫹ channels.
Instead, Ca2⫹ oscillations result from pulsed influxes of
Ca2⫹ through short-lived HlyA pores, which are rapidly
closed or removed from the membrane. The causal
connection was directly seen in simultaneous patchclamp measurements of pore formation and Ca2⫹
measurements, where Ca2⫹ elevations occurred as a
consequence of opening of HlyA pores.
Pore formation by HlyA happens at random, which
explains our observation that the Ca2⫹ peaks in individual cells occur in a stochastic sequence. This contrasts a previous claim on the existence of periodic
low-frequency Ca2⫹ oscillations induced by HlyA. However, the mathematical model that this claim was based
on turned out as inadequate for a proper interpretation
of our measured Ca2⫹ data. Moreover, we found that
the mean time span between two Ca2⫹ elevations was
dependent on the concentration of HlyA, whereupon
WHY E. COLI ␣-HEMOLYSIN INDUCES CALCIUM OSCILLATIONS
higher concentrations led to shorter time spans. This
concentration dependence definitely rules out the idea
that HlyA molecules may switch on in the target cell a
periodic “Ca2⫹ oscillation run” with one defined frequency.
Washout experiments showed that pore formation,
propidium iodide influx, and Ca2⫹ oscillations only
occurred as long as HlyA molecules were present at the
extracellular side. The observation that the Ca2⫹ oscillations ceased with the start of washout demonstrates
further that the cell has no lasting memory on the
encounter with HlyA regarding [Ca2⫹]i.
Whensoever Ca2⫹ oscillations are initiated by HlyA,
the pore is on its own. Nonperiodic (“chaotic”) Ca2⫹
oscillations resulting from the interplay of formation
and disappearance of the pores along with cellular
Ca2⫹ redistribution will obviously vary depending on
toxin concentration and susceptibility and may provide
the starting point of myriad reactions, but on a very
individual basis, in cells attacked by pore-forming toxins such as HlyA.
975
The FASEB Journal • FJ Express Full-Length Article
Why Escherichia coli ␣-hemolysin induces calcium
oscillations in mammalian cells—the pore is
on its own
¨ nver,* Florian Dreyer,*
Andreas Koschinski,*,1 Holger Repp,*,1,2 Baris U
†
Dierk Brockmeier,* Angela Valeva, Sucharit Bhakdi,† and Iwan Walev†,2
*Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University, Giessen, Germany; and
†
Institute of Medical Microbiology and Hygiene, Johannes-Gutenberg-University, Mainz, Germany
Escherichia coli ␣-hemolysin (HlyA), archetype of a bacterial pore-forming toxin, has been
reported to deregulate physiological Ca2ⴙ channels,
thus inducing periodic low-frequency Ca2ⴙ oscillations
that trigger transcriptional processes in mammalian
cells. The present study was undertaken to delineate
the mechanisms underlying the Ca2ⴙ oscillations.
Patch-clamp experiments were combined with single
cell measurements of intracellular Ca2ⴙ and with flowcytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca2ⴙ oscillations in human renal and endothelial cells. However, contrary to
the previous report, the phenomenon could not be
inhibited by the Ca2ⴙ channel blocker nifedipine and
Ca2ⴙ oscillations showed no constant periodicity at all.
Ca2ⴙ oscillations were dependent on the pore-forming
activity of HlyA: application of a nonhemolytic but
bindable toxin had no effect. Washout experiments
revealed that Ca2ⴙ oscillations could not be maintained
in the absence of toxin in the medium. Analogously,
propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable
toward the dye after toxin washout, indicating resealing
or removal of the membrane lesions. Finally, patchclamp experiments revealed temporal congruence between pore formation and Ca2ⴙ influx. We conclude
that the nonperiodic Ca2ⴙ oscillations induced by HlyA
are not due to deregulation of physiological Ca2ⴙ
channels but derive from pulsed influxes of Ca2ⴙ as a
consequence of formation and rapid closure of HlyA
pores in mammalian cell membranes.—Koschinski, A.,
¨ nver, B., Dreyer, F., Brockmeier, D., ValRepp, H., U
eva, A., Bhakdi, S., and Walev, I. Why Escherichia coli
␣-hemolysin induces calcium oscillations in mammalian
cells—the pore is on its own. FASEB J. 20, E80 –E87
(2006)
ABSTRACT
Key Words: pore-forming toxins 䡠 RTX toxins 䡠 gram-negative
bacteria 䡠 membrane lesions
ESCHERICHIA COLI ␣-hemolysin (HlyA) is prototypic of a
large family of pore-forming toxins that are produced
by many medically important gram-negative pathogens.
E80
Common to all are the presence of a Ca2⫹ binding
nonapeptide repeat sequence and the requirement for
posttranslational fatty acylation at one or two lysine
residues for the acquisition of pore-forming activity
(1, 2). In the absence of Ca2⫹ or fatty acylation, binding
capacity is retained but the toxins are unable to adopt
the pore-forming configuration (3, 4). The question of
whether HlyA binds to specific cellular receptors is
currently under investigation (5, 6); the toxin binds to
protein-free lipid bilayers to form pores of ⬃1 nm
diameter (7), similar to the pore size described in
animal cell membranes (8). Pores formed in planar
lipid bilayers flicker between an open and closed state
dependent on the membrane potential (7, 9).
In addition to their direct cytotoxic capacity, poreforming toxins can trigger a multitude of cellular
responses that may in turn produce important longrange effects in the mammalian host organism. Many
reactions are triggered by the uncontrolled flux of
monovalent and divalent ions across the plasma membrane (10). Studies with S. aureus alpha-toxin and
streptolysin O have disclosed that nucleated cells are
able to repair a limited number of lesions (11, 12) and
that this is accompanied by transcriptional responses
such as the activation of NF-␬B and subsequent cytokine production (13, 14). Thus, similar to endotoxin,
pore-forming toxins may provoke a large spectrum of
long-term effects, and delineating the mechanisms underlying transcriptional activation represents a novel
field of research with broad potential relevance.
Oscillations in the cytoplasmic concentration of free
Ca2⫹ have recently been recognized to represent a
central event in transcriptional regulation (15, 16).
Therefore, the finding that subcytocidal attack by HlyA
is accompanied by periodic low-frequency Ca2⫹ oscilla1
These authors contributed equally to this work.
Correspondence: Frankfurter Str. 107, Rudolf-BuchheimInstitute of Pharmacology, Justus-Liebig-University, 35392
Giessen, Germany. Email: Holger.Repp@pharma.med.unigiessen.de; and Obere Zahlbacher Str. 67, Institute of Medical
Microbiology and Hygiene, Johannes-Gutenberg-University,
55101 Mainz, Germany. Email: walev@mail.uni-mainz.de
doi: 10.1096/fj.05-4561fje
2
0892-6638/06/0020-0080 © FASEB
tions, transcriptional activation, and interleukin-6 production in renal epithelial cells represented an important discovery that might be extrapolatable to other
pore-forming agents (17). The question relating to the
cause of the HlyA-induced periodic Ca2⫹ oscillations
also appeared to be resolved in that publication: Ca2⫹
oscillations were reportedly suppressed in the presence
of the Ca2⫹ channel blocker nifedipine. Thus, in
addition to being a pore-forming toxin, HlyA appeared
to be endowed with the capacity of influencing physiological Ca2⫹ channels in mammalian cells (17). The
present investigation was undertaken to make a closer
investigation of this putative bifunctionality of HlyA.
The capacity of the toxin to induce Ca2⫹ oscillations
in mammalian cells was confirmed. However, Ca2⫹
oscillations showed no constant periodicity at all. Furthermore, a different conclusion was reached regarding the cause of Ca2⫹ oscillations. Evidence is presented that they are not due to toxin-dependent
alterations of intrinsic Ca2⫹ channel activity but to
pulses of Ca2⫹ flux through short-lived toxin pores,
which are rapidly closed or removed from the plasma
membrane of target cells.
MATERIALS AND METHODS
Cell culture
starting with the mutant K564R/K690R to form S177C/
K564R/K690R. Mutant toxins containing Cys177 (S177C and
S177C/K564R/K690R) were labeled with [3H]N-ethyl-maleimide. The binding capacity of these labeled toxins to erythrocytes was determined. The bindability of labeled mutant
S177C and the hemolytically inactive mutant S177C/K564R/
K690R was identical. Protein purification of toxins was carried
out as described previously (1,4).
Electrophysiological recording
Cells were plated in 35 mm dishes 48 h before the experiments. Cells (⬃4⫻105 per dish) were washed with extracellular (bath) solution (in mM: 140 NaCl, 3 KCl, 2 MgCl2, 2
CaCl2, 10 glucose, and 10 HEPES, pH 7.35 adjusted with ⬃4
mM NaOH), and whole-cell recordings were started within
5–10 min after the washing procedure, using a bath volume of
2 ml. Recording pipettes had a resistance of 5–10 M⍀ when
filled with pipette solution (in mM: 140 K⫹ glutamate, 10
NaCl, 2 MgCl2, and 10 HEPES, pH 7.3 adjusted with ⬃4 mM
KOH). A free intracellular Ca2⫹ concentration ([Ca2⫹]i) of
100 nm was obtained using 100 ␮M of the Ca2⫹ chelator
BAPTA and a total Ca2⫹ concentration of 30 ␮M, assuming
an apparent dissociation constant Kd of 0.24 ␮M (pH 7.3) for
the Ca2⫹ BAPTA complex. Solutions were filtered through
0.2 ␮m pore size filters. Whole-cell current recording and
data analysis were performed as described previously (18,19).
HlyA was applied with a pipette directly into the bath solution
within 2–5 min after a stable recording configuration had
been obtained. All measurements were performed at 20 –
22°C. Data are mean ⫾ sem unless stated otherwise.
Measurement of intracellular ATP
Human embryonic kidney (HEK293) cells were maintained
in a humidified, 6% CO2-94% air atmosphere in a mixture of
Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s
F-12 medium (1:1, v/v) containing 10% (v/v) fetal calf serum
(PAN Systems, Aidenbach, Germany) and 2 mM l-glutamine
without antibiotics. The human endothelial cell line EAhy926
(a kind gift of Cora-Jean S. Edgell, University of North
Carolina, Chapel Hill, NC), a hybridoma derived from the
fusion of human umbilical vein endothelial cells with the
A549 cell line, was grown in DMEM/Nutrient Mix F-12 (1:1)
with Glutamax I and pyrodoxin (Life Technologies, Paisley,
Scotland) supplemented with 10% fetal calf serum (Life
Technologies), 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Life Technologies) at 37°C in humidified air containing 6% CO2. Human renal proximal tubule-derived cells
(IHKE) were kindly provided by Dr. Michael Gekle (Institute
of Physiology, University of Wu¨rzburg, Wu¨rzburg, Germany).
The cells were originally from Dr. Steen Mollerup (Department of Toxicology, National Institute of Occupational
Health, Oslo, Norway) who kindly permitted us to use them.
Cells were grown in Ham’s F-12 modified DMEM medium
supplemented with 1.18 g/l NaHCO3, 5.37 g/l HEPES, 5
mg/l human apo-transferrin, 5 mg/l bovine insulin, 36 ␮g/l
hydrocortisone, 10 ␮g/l mouse epidermal growth factor, 5
␮g/l Na-selenite, and 10% fetal calf serum. Cells were grown
in a 37°C, 94% air-6% CO2 incubator.
Expression of HlyA, mutagenesis, and toxin purification
Hemolytically inactive mutant in which Lys at position 564
and 690 was replaced by Arg (K564R/K690R) (1) was kindly
provided by C. Hughes and V. Koronakis (Department of
Pathology, Cambridge University, Cambridge, UK). Mutation
of Ser-177 into Cys in cysteine-less wild-type (WT) HlyA was
described previously (4). The same procedure was followed
WHY HlyA INDUCES CALCIUM OSCILLATIONS
HEK293 cells in 96-well plates were treated with various
concentrations of HlyA for 60 min, after which the supernatants were removed. Cellular ATP concentrations were determined after the cells were lysed with 0.2 ml of 0.5% (v/v)
Triton X-100, using a commercial ATP-bioluminescence kit
(Boehringer Mannheim). Intracellular ATP was expressed as
the percentage of luminescence relative to that of negative
controls (without toxin).
Ratiometric Ca2ⴙ imaging
Cells were plated on 35 mm plastic petri dishes that had been
modified for fluorescence measurements (18). Cells were
loaded for 30 min at 37°C with the fluorescent Ca2⫹ indicator
Fura 2-acetoxymethyl ester (AM) (Calbiochem-Novabiochem,
Bad-Soden, Germany) dissolved in extracellular solution at a
concentration of 5 ␮M. The solution contained 16 ␮M
Pluronic F-127 (Molecular Probes, Eugene, OR) for a better
dispersion of Fura 2-AM. Loaded cells were washed extensively with extracellular solution and further incubated for 10
min at 37°C. Measurements of [Ca2⫹]i were performed at
room temperature with a Leica DM IRB inverse microscope
and a VisiChrome imaging system (Visitron, Puchheim, Germany). Excitation was at 350 and 380 nm, and emission was
determined at 510 nm. [Ca2⫹]i was determined ratiometrically from the captured data using the Metafluor imaging
software.
Biometric analysis
A time period of 25.55 min (512 measurements, sampling
interval 3 s) of each individual original Ca2⫹ tracing was
subjected to a discrete Fourier transformation, followed by
E81
computation of the respective power spectrum. According to
the sampling period (T⫽25.55 min), multifolds of the basic
angular velocity (␻0⫽2␲/T) constitute the Fourier transformation, corresponding to wavelengths of 25.55, 12.775, 8.517,
6.388, etc., min. In addition, the signals of these data sets were
first corrected by possible baseline drifts using a trend function that consisted in a second order polynomial function,
i.e., a constant, a linear, and a quadratic term. The trend
function was estimated by a Gaussian least square method.
After subtraction of the trend, the signals were treated as
described above. No further data manipulations or standardization was carried out.
trace. As a negative membrane holding potential of –50
mV was used, the membrane current trace steps down
during a pore opening and up during a pore closing.
Thus, Fig. 1A also demonstrates that the HlyA pores did
not remain continuously open but oscillated between
open and closed states (see also Figs. 4B and 5B and C).
Despite ongoing pore formation, the cells remained
viable when toxin concentrations of ⱕ10 ng/ml were
employed, and no decreases in cellular ATP levels were
noted (Fig. 1B).
Online supplemental material
HlyA induces nonperiodic Ca2ⴙ oscillations
in renal cells
Figure S1 is a time-lapse video of monitoring of [Ca2⫹]i in
IHKE cells after exposure to HlyA (2.5 ng/ml). The original
recording period was 90 min. Figure S2 shows Ca2⫹ tracings
of 52 different IHKE cells after exposure to HlyA (2.5 ng/ml).
Figure S3 shows a time-lapse video of [Ca2⫹]i in IHKE cells
after exposure to HlyA (twice 2.5 ng/ml) in the presence of
nifedipine. Ca2⫹ oscillations become visible after the change
of the field of view. Figure S4 is a time-lapse video of the effect
of HlyA (2.5 ng/ml) on [Ca2⫹]i in serum-starved (24 h) IHKE
cells. Figure S5 shows Ca2⫹ tracings of 30 different serumstarved (24 h) IHKE cells exposed to HlyA (2.5 ng/ml).
RESULTS
Formation of HlyA pores in HEK293 cells
HEK293 cells were initially used to characterize the
pore-forming effects of HlyA in the whole-cell configuration of the patch-clamp technique. HEK293 cells
possess only low endogenous ion channel activity at
certain membrane potentials (20) and are well suited
for electrophysiological experiments to measure the
effect of pore-forming toxins. This cell type already has
been successfully used in our group to study pore
formation by listeriolysin of Listeria monocytogenes (18).
Pore formation was observed within seconds after application of HlyA and followed a steep concentrationresponse curve (Fig. 1A). At a concentration of 5 ng/ml
HlyA, ⬃230 s elapsed from the opening of the first pore
to the opening of the tenth pore. A 10-fold higher
concentration reduced this time span to ⬃40 s. With
the patch-clamp-technique, a determination of single
pores is possible, since each pore opening becomes
visible by a stepwise change in the membrane current
IHKE cells were loaded with Fura 2-AM and used to
study the effect of HlyA on [Ca2⫹]i. IHKE cells were
chosen as human equivalent to primary rat proximal
tubule (RPT) cells that were used in a previous study to
demonstrate the effect of HlyA on [Ca2⫹]i (17). IHKE
cells are easily accessible for electrophysiological and
Ca2⫹ measurements. Within ⬃30 s after application of
5 ng/ml HlyA to the cells, [Ca2⫹]i increased and
exhibited oscillations during further monitoring (Fig.
2A-E). It was previously reported that HlyA provoked
periodic Ca2⫹ oscillations in RPT cells, with a constant
periodicity of Ca2⫹ peaks of 12 min (17). However, we
found large variations of oscillation kinetics and peakto-peak spans in human tubule cells. Figure 2A-D shows
Ca2⫹ tracings of four individual cells after application
of HlyA, already giving an impression of the broad
variability of the Ca2⫹ oscillations. False-color images of
[Ca2⫹]i in individual cells are demonstrated in Fig. 2E.
A time-lapse video of Ca2⫹ monitoring of IHKE cells is
available online (Fig. S1), as well as Ca2⫹ tracings of
⬃50 different IHKE cells (Fig. S2), demonstrating
impressively that the Ca2⫹ oscillations showed no constant periodicities of the peaks and large variations of
the peak-to-peak spans. We then applied the identical
mathematical method that was previously used to identify HlyA-induced periodic Ca2⫹ oscillations of low
frequency (17), i.e., a power spectrum analysis that is
based on a discrete Fourier transformation. Despite the
marked heterogeneity of the original Ca2⫹ tracings, the
analysis of the data sets yielded a periodicity of Ca2⫹
peaks with a dominant wavelength of 12.8 min, which
was in remarkable agreement with the previously reported value of 12.0 min in RPT cells (17). As a control,
Figure 1. A) Pore-forming activity of HlyA in
HEK293 cells. Relationship of toxin concentration and
time between opening of the first and the tenth pore
(⌬tp1–10). Insets show courses of pore formation in
single cells after exposure to (1) 50 ng/ml and (2) 6.25
ng/ml HlyA. Trace goes downward after a pore opening and upward during a pore closing. Membrane
holding potential was –50 mV. This negative potential
avoids activation of endogenous Ca2⫹ and K⫹ channels
in HEK293 cells. B) Effect of HlyA on intracellular ATP
concentration of HEK293 cells. Cells were exposed to
HlyA at given concentrations for 60 min, 37°C, and
cellular ATP was determined. Error bars indicate sd.
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Vol. 20
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The FASEB Journal
KOSCHINSKI ET AL.
Figure 2. Ca2⫹ oscillations induced by HlyA in
IHKE cells loaded with the fluorescent Ca2⫹
indicator Fura 2-AM. A–D) Cells were exposed
to HlyA (5 ng/ml) 3 min after start of recording. Traces represent examples of time courses
of [Ca2⫹]i of 4 single cells. E) Single frames
from an image series of [Ca2⫹]i. Frames 1– 4
correspond to numbers 1– 4 above trace in A.
Traces in A and B were recorded from cells that
are marked with A and B. F) Superposition of
Ca2⫹ responses of 49 IHKE cells in the presence
of nifedipine (100 ␮M). Application of HlyA (5
ng/ml) is indicated by arrow. Gap represents
time when field of view was changed to an area
that had not been UV irradiated before. G)
Ca2⫹ tracing of a single IHKE cell on consecutive exposure to a nonhemolytic HlyA mutant
(⫽nh HlyA, 25 ng/ml) and WT HlyA (2.5
ng/ml).
tracings monitored in the absence of HlyA were also
analyzed. Surprisingly, a periodicity of peaks with a
wavelength of ⬃12 min was again computed, although
no Ca2⫹ elevations existed in the control tracings.
Furthermore, analysis of a signal consisting only of
white noise superimposed to a discretely drifting baseline yielded the same peak value. Thus, it became clear
that this mathematical model was not adequate for a
proper interpretation of the measured Ca2⫹ data. Indeed, due to the properties of discrete Fourier transformation, the power spectrum will provide information for discrete frequencies only, depending on the
sampling period and sampling frequency. In the experimental constellation we used, which is comparable to
the constellation described in the previous report (17),
the power spectrum is computed for wavelengths of
25.5, 12.8, 8.5, etc., min. The discrete Fourier transformation power spectrum analysis is not suitable to trace
oscillations for these low frequencies, since variations in
baseline do produce prominent peaks at the same
discrete frequencies, which can lead to a fundamental
misinterpretation of measured data.
We next determined the statistical distribution of the
time spans between directly neighboring Ca2⫹ peaks in
Fig. 2A-D and 48 other Ca2⫹ tracings. Figure 3 shows
that the most frequent peak-to-peak span was 70 s and
that spans in the range of 1–3 min occurred more
frequently compared to longer time spans, whereas
WHY HlyA INDUCES CALCIUM OSCILLATIONS
peak-to-peak spans above 10 min were very rare events.
Importantly, these peak-to-peak spans represent the
time span between a Ca2 ⫹peak to its direct neighbor.
They should not be confused with peak periodicity or
frequency of consecutive peaks. When the toxin concentration was increased, the peak-to-peak span distribution shifted to shorter time spans, as illustrated in
Fig. 3, inset, by a typical Ca2⫹ tracing where two
different HlyA concentrations were applied consecu-
Figure 3. Frequency distribution of time spans between
consecutive Ca2⫹ peaks in Ca2⫹ tracings monitored in 52
IHKE cells. Cells were loaded with Fura 2-AM and exposed to
HlyA (5 ng/ml). Analysis period was 55 min. Curve was fitted
to data using weighted average of 9 nearest neighbors. Bin
width was 10 s. Inset shows Ca2⫹ monitoring of a single IHKE
cell in the presence of 2.5 and 7.5 ng/ml HlyA.
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tively. The finding that the peak-to-peak span distribution is dependent on HlyA concentration clearly refutes
the idea that the HlyA molecule might have the inherent feature to induce Ca2⫹ oscillations of a discrete
frequency.
To address the question of a possible cell-type limitation of our observations, we further investigated the
Ca2⫹ response to HlyA in HEK293 and EAhy926 cells.
The EAhy926 line is representative of human vascular
endothelial cells and was used to have a complement to
IHKE cells, which have an epithelial nature. In HEK293
and EAhy926 cells, Ca2⫹ oscillations were also observed
but with no evidence of a constant periodicity (data not
shown).
The results presented yet were obtained with nonsynchronized cells. Thus, one might speculate that the
observed nonperiodic Ca2⫹ oscillations could be cellcycle dependent. To address this question, IHKE cells
were subjected to 12–24 h serum starvation, and then
Ca2⫹ measurements were performed. Despite synchronization of the cell cycle, HlyA (2.5 ng/ml)-induced
Ca2⫹ oscillations were still nonperiodic, and no synchronization of Ca2⫹ elevations was observable. A timelapse video of Ca2⫹ monitoring of serum-starved IHKE
cells is available online (Fig. S4), as well as 30 individual
Ca2⫹ tracings of serum-starved IHKE cells (Fig. S5).
These data clearly show that also in synchronized cells,
the Ca2⫹ response to HlyA is nonperiodic and not
synchronous.
Nifedipine does not inhibit Ca2ⴙ oscillations
When voltage-gated L-type Ca2⫹ channels in IHKE cells
were blocked by nifedipine (100 ␮M), Ca2⫹ oscillations
were still observable after application of HlyA (Fig. 2F,
Fig. S3). This finding contrasted with the observations
in RPT cells where nifedipine (100 ␮M) abolished the
Ca2⫹ response to HlyA (17). A striking phenomenon
that we observed during Ca2⫹ measurements in the
presence of nifedipine can account for this difference.
Those cells that were in the microscopic field of view
for several minutes, i.e., irradiated by ultraviolet (UV)
light, did indeed not show Ca2⫹ oscillations after
application of HlyA (Fig. 2F, Fig. S3). Once adjusted,
the field of view is usually not changed during the
further Ca2⫹-monitoring period. However, when, after
application of HlyA, the field of view was changed to
cells that had not been in the optical path before, i.e.,
had not “seen” UV light, Ca2⫹ oscillations were observed (Fig. 2F, Fig. S3). This phenomenon was reproduced in three independent experiments in IHKE cells
and also in HEK293 and EAhy926 cells. Thus, the
previously observed elimination of HlyA-induced Ca2⫹
oscillations by nifedipine was essentially an experimental artifact.
Additionally, HlyA still led to Ca2⫹ oscillations in
HEK293 cells when a combination of Ca2⫹ channel
blockers was present, including nifedipine (100 ␮M);
Cd2⫹ (2 mM), which blocks all types of voltage-gated
Ca2⫹ channels (21); and SK&F 96365 (25 ␮M), a
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May 2006
blocker of receptor-operated Ca2⫹ channels (22). This
shows that the effect of HlyA on [Ca2⫹]i is not dependent on the activity of endogenous membrane Ca2⫹
channels.
Pore formation and Ca2ⴙ oscillations do not occur in
cells treated with nonhemolytic HlyA
IHKE and HEK293 cells were treated with the HlyA
mutant S177C/K564R/K690R, which is nonhemolytic
but fully bindable. In no case were pore formation and
Ca2⫹ oscillations ever noted at concentrations of up to
500 ng/ml. An example of the course of [Ca2⫹]i in
IHKE cells with consecutive exposure to nonhemolytic
HlyA mutant and to WT HlyA is shown in Fig. 2G. These
results demonstrated that the ability of HlyA to provoke
Ca2⫹ oscillations was not related simply to binding to
the plasma membrane but required formation of pores.
Pore formation and Ca2ⴙ oscillations cease rapidly
on removal of HlyA
We then addressed the question of whether the effect
of HlyA on [Ca2⫹]i persists after removal of the toxin
from the extracellular medium. Figure 4A shows a
superposition of the Ca2⫹ responses of 31 IHKE cells
after application of HlyA (10 ng/ml). Shortly after the
start of a bath perfusion to wash out HlyA from the
extracellular medium, [Ca2⫹]i decreased and reached
the initial concentration after ⬃3 min. A second application of HlyA (5 ng/ml) again led to Ca2⫹ oscillations,
which disappeared within some minutes after restart of
the bath perfusion. The disappearance of the Ca2⫹
oscillations after washout of HlyA followed the same
time course as observed for the disappearance of pore
formation. Figure 4B shows a typical whole-cell registration of pore formation in a single IHKE cell after
application of HlyA. Open pores were no longer observed ⬃4 min after the start of the bath perfusion.
Reapplication of HlyA again led to pore formation.
The most straightforward explanation for the above
findings was that after formation, the HlyA pores
rapidly disappeared from the plasma membrane, either
through closure or by removal from the afflicted membrane area. To corroborate this, influx of propidium
iodide was investigated in parallel. As shown in Fig.
4C-E, cells became permeable to the dye during incubation with HlyA, but membranes returned to the
impermeable state shortly after toxin removal. This
confirmed that pores formed by HlyA were short lived.
Evidence that Ca2ⴙ oscillations result from Ca2ⴙ
influx through HlyA pores followed by Ca2ⴙ
redistribution
We then simultaneously monitored pore formation by
HlyA (10 ng/ml) and [Ca2⫹]i in IHKE cells. We found
an increase in [Ca2⫹]i directly after pore opening and
a good temporal correlation between formation and
closure of HlyA pores and Ca2⫹ elevations (Fig. 5A, B).
The FASEB Journal
KOSCHINSKI ET AL.
Figure 4. A) Superposition of the Ca2⫹ responses of 31 IHKE cells after exposure to HlyA
(10 ng/ml), followed by a washout, and a
repeat of that sequence with 5 ng/ml HlyA. B)
Monitoring of pore formation in a single
HEK293 cell after application of HlyA (10 ng/
ml), followed by superfusion of the bath chamber with extracellular solution to wash out the
toxin, and second application of HlyA (10
ng/ml). Insets 1 and 2 show pore formation at
a higher time resolution, revealing that pores
did not remain continuously open but oscillated between open and closed states. Membrane holding potential was –50 mV. Membrane current trace goes downward during a
pore opening and upward during closing. C–E)
Demonstration that IHKE cells are permeable
for propidium iodide (PI) dependent on the
presence of HlyA in the medium. C) Cells
exposed to PI alone. D) PI was added to the cells
together with 50 ng/ml HlyA. E) PI was added
after HlyA removal. After 15 min incubation
with PI, samples were subjected to flow cytometric analysis. Cells simultaneously exposed to
HlyA and PI showed red fluorescence because
of flux of PI into the cells. In contrast, cells
exposed to PI after removal of HlyA were not
stained.
We hypothesized that the decrease after an elevation
might reflect the restoration of Ca2⫹ homeostasis by
physiological Ca2⫹ redistribution after HlyA pore closure. To test this possibility, IHKE cells were incubated
with thapsigargin (1 ␮M), an inhibitor of endoplasmic
reticular Ca2⫹ ATPases. We found that the effect of
thapsigargin depended on the concentration of HlyA.
In the presence of thapsigargin and 2.5 ng/ml HlyA,
most IHKE cells showed typical Ca2⫹ oscillations. In the
presence of thapsigargin and 5 ng/ml HlyA, some
IHKE cells showed typical Ca2⫹ oscillations even during
several hours of monitoring. The major part of the cells
exhibited Ca2⫹ oscillations only for a limited period of
⬃30 min, and thereafter a continuous increase in [Ca2⫹]i
was observed. Some cells showed no Ca2⫹ oscillations but
only a continuous elevation in [Ca2⫹]i. When thapsigargin-treated IHKE cells were exposed to 10 ng/ml HlyA,
most cells responded with a continuous increase in
[Ca2⫹]i. In IHKE cells that were not treated with thapsigargin, HlyA concentrations of at least 20 ng/ml were
necessary to induce a continuous elevation in [Ca2⫹]i.
DISCUSSION
The results of this study led us to a new explanation for
Ca2⫹ oscillations that occur in mammalian cells treated
WHY HlyA INDUCES CALCIUM OSCILLATIONS
with HlyA. Three human cell-types were used for this
investigation, namely renal embryonic, renal tubule
epithelial, and vascular endothelial cells. The Ca2⫹
oscillations are not thought to be due to any direct or
indirect influence of HlyA on intrinsic Ca2⫹ channels.
Instead, they are proposed to result from pulsed influxes of Ca2⫹ through short-lived toxin pores, which
we believe are rapidly sealed or removed from the
membrane. Given the fundamental relevance of cytoplasmic Ca2⫹ oscillations for many cell functions, this
conceptual remodeling will be of importance for future
investigations in the field. Arguments in support of the
proposal are manifold.
First, we were unable to confirm the suppressive
effect of the Ca2⫹ channel blocker nifedipine on HlyAinduced Ca2⫹ oscillations in any of the three cell-types
studied. It is known that nifedipine is very sensitive to
UV and daylight (up to 450 nm). It is readily converted
into degradation products, which are no longer active
against Ca2⫹ channels but instead have cellular as well
as toxic effects (23–25). The degradation products
might lead to cell damage, with the consequence of
missing Ca2⫹ oscillations in UV-irradiated cells. In
conclusion, the previously observed elimination of
HlyA-induced Ca2⫹ oscillations by nifedipine was essentially not due to Ca2⫹ channel blockade, and the sole
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Figure 5. A, B) Parallel monitoring of [Ca2⫹]i and pore
formation by HlyA (10 ng/ml) in a single IHKE cell. A) Ca2⫹
concentration trace. B) Electrophysiological recording of
pore formation. Solid line in B is an inverse display of the
Ca2⫹ concentration trace shown in A for a direct comparison
of [Ca2⫹]i and pore formation. C) Part of the trace shown in
B but at a higher time resolution for a better discrimination of
pore openings and closings. Membrane holding potential was
–50 mV. Membrane current trace goes downward during a
pore opening and upward during closing.
support for the postulated crucial involvement of intrinsic L-type Ca2⫹ channels in HlyA-provoked Ca2⫹
oscillations (17) ceased to exist. Furthermore, we found
that the effect of HlyA on [Ca2⫹]i is not dependent on
the activity of any voltage-gated or receptor-operated
membrane Ca2⫹ channels.
Secondly, washout experiments provided an important further clue. Removal of HlyA from the medium
resulted in rapid disappearance of the pores. At the
same time, the ability of propidium iodide to diffuse
into the cells was lost and, strikingly, Ca2⫹ oscillations
also ceased. The conclusion was rather inescapable
that, after formation in mammalian cell membranes,
HlyA pores are short lived and disappear within seconds, either by rapid closure or by removal from the
membrane.
Final support for the pore theory of Ca2⫹ oscillations
came from simultaneous patch-clamp measurements of
pore formation and Ca2⫹ measurements. These experiments directly showed that Ca2⫹ elevations occurred as
a consequence of opening of the pores formed by HlyA.
Pore openings occur at random, and the open probability depends on HlyA concentration. Since a rise in
[Ca2⫹]i is an effect that is directly conditioned by a
pore opening, it is also expected to happen stochastically, and the probability should be dependent on toxin
concentration. In contrast to a previous report (17), we
found indeed no evidence for the existence of a
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May 2006
constant periodicity of the Ca2⫹ peaks, and we observed
that the time spans between consecutive Ca2⫹ peaks
depended on HlyA concentration. This concentration
dependence definitely rules out the idea that HlyA
molecules may switch on in the target cell a periodic
“Ca2⫹ oscillation run” with one defined frequency.
The concept thus took shape that Ca2⫹ fluxed from
the extracellular compartment through the stochastically opened HlyA pores to the cytosol and that this was
followed by pore closure and a rapid decline of the
elevated cytoplasmic Ca2⫹ concentration, indicating
restoration of cellular Ca2⫹ homeostasis. This concept
is supported by the observed dependence of the Ca2⫹
response on HlyA concentration in IHKE cells treated
with thapsigargin. It appears that cells respond to Ca2⫹
influx through HlyA pores with uptake into intracellular Ca2⫹ stores combined with discharge by plasma
membrane Ca2⫹ pumps. If a part of these systems is cut
off, e.g., by thapsigargin, the amount of Ca2⫹ influx
determines whether Ca2⫹ homeostasis can still be maintained over time or not. As the amount of Ca2⫹ influx
depends on the number of HlyA pores, it can be well
explained that the effect of thapsigargin is dependent
on HlyA concentration.
In summary, a novel concept has emerged to account
for the intriguing finding that a pore-forming toxin can
provoke Ca2⫹ oscillations in living cells. The experiments described herein provide evidence that an RTX
toxin forms very short-lived pores in cell membranes. In
fact, similar rapid pore closure has been noted in
artificial lipid bilayers (7–9). Thus, the very short pore
life may be an intrinsic property that is governed by the
target bilayer itself. Nonperiodic (“chaotic”) Ca2⫹ oscillations resulting from the interplay of formation and
disappearance of the pores along with cellular Ca2⫹
redistribution will obviously vary depending on toxin
concentration and susceptibility and may provide the
starting point of myriad reactions, but on a very individual basis, in cells attacked by pore-forming toxins
such as HlyA.
We thank C. Zibuschka and S. Weis for expert technical
assistance. This work was supported by grants from the
Deutsche Forschungsgemeinschaft (RE 1046/1–2 and SFB
490, project C1/D3).
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Received for publication August 18, 2005.
Accepted for publication January 3, 2006.
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