Cellular Microbiology (2002) 4(8), 483–491
Listeriolysin of Listeria monocytogenes forms
Ca2+-permeable pores leading to intracellular
Ca2+ oscillations
Holger Repp,1 Zübeyde Pamukçi,1
Andreas Koschinski,1 Eugen Domann,2 Ayub Darji,2
Jan Birringer,1 Dierk Brockmeier,1
Trinad Chakraborty2* and Florian Dreyer1*
1
Rudolf-Buchheim-Institute of Pharmacology and
2
Institute of Medical Microbiology, Faculty of Medicine,
Justus-Liebig-University of Giessen, Frankfurter Str.
107, 35392 Giessen, Germany.
Summary
Listeriolysin (LLO) is a major virulence factor of
Listeria monocytogenes, a Gram-positive bacterium
that can cause life-threatening diseases. Various signalling events and cellular effects, including modulation of gene expression, are triggered by LLO through
unknown mechanisms. Here, we demonstrate that
LLO applied extracellularly at sublytic concentrations
causes long-lasting oscillations of the intracellular
Ca2+ level of human embryonic kidney cells; resulting
from a pulsed influx of extracellular Ca2+ through
pores that are formed by LLO in the plasma membrane. Calcium influx does not require the activity of
endogenous Ca2+ channels. LLO-formed pores are
transient and oscillate between open and closed
states. Pore formation and Ca2+ oscillations were also
observed after exposure of cells to native Listeria
monocytogenes. Our data identify LLO as a tool
used by Listeria monocytogenes to manipulate the
intracellular Ca2+ level without direct contact of the
bacterium with the target cell. As Ca2+ oscillations
modulate cellular signalling and gene expression, our
findings provide a potential molecular basis for the
broad spectrum of Ca2+-dependent cellular responses
induced by LLO during Listeria infection.
Introduction
Listeria monocytogenes is a food-borne, Gram-positive
Received 8 March, 2002; revised 6 May, 2002; accepted 10
May, 2002 *For correspondence. E-mail Trinad.Chakraborty@
mikrobio.med.uni-giessen.de; Tel. (+49) 6419941280; Fax: (+49)
6419941259; E-mail
[email protected];
Tel. (+49) 6419947620; Fax (+49) 6419947609.
© 2002 Blackwell Science Ltd
pathogen that can cause life-threatening diseases
such as meningitis, meningoencephalitis, septicaemia
and severe gastroenteritis (Schuchat et al., 1991) with an
overall mortality rate of more than 20% (Lorber, 1997).
Natural infection with L. monocytogenes may occur after
consumption of Listeria-contaminated food such as soft
cheese and raw milk (Farber and Peterkin, 1991). Infants,
pregnant women, the elderly and immunocompromised
individuals are especially susceptible. During disseminated systemic infections, many organ systems and
cell types are affected and many of the virulence factors
produced by the bacterium are involved in sustaining
its spread and survival (Portnoy et al., 1992; Tilney and
Tilney, 1993; Cossart and Lecuit, 1998; Chakraborty,
1999; Vázquez-Boland et al., 2001).
A major virulence factor of L. monocytogenes is LLO
(Portnoy et al., 1988; Cossart et al., 1989), a toxin belonging to the family of cholesterol-dependent, pore-forming
cytolysins (CDTX). Other members of this family include
streptolysin, perfringolysin, pneumolysin and alveolysin,
all of which are secreted products of Gram-positive
pathogens. Currently, this group is composed of 23
members with a considerable degree of similarity
(40–70%) at the protein level (Alouf, 2000; Tweten et
al., 2001). Despite its structural similarities to other
CDTX members, LLO exhibits unique virulence properties
(Portnoy et al., 1992).
LLO is a sine qua non of listerial infections, and strains
lacking this gene are rendered non-pathogenic. During
infection, LLO has been shown to be required at a multitude of steps in the intracellular life cycle of L. monocytogenes, such as in modulating internalization, escape
from the vacuole, proliferation in the cytoplasm, and
cell-to-cell spread (Portnoy et al., 1992; Tilney and
Tilney, 1993; Cossart and Lecuit, 1998; Chakraborty, 1999;
Vázquez-Boland et al., 2001). In addition, a plethora of
cellular responses to LLO has been reported, including
activation of the MAP kinase signalling pathway (Tang et
al., 1996), production of host signalling molecules such as
inositol trisphosphate and diacylglycerol (Sibelius et al.,
1996), modulation of cytokine gene expression (Nishibori
et al., 1996; Kohda et al., 2002), expression of cell adhesion molecules on infected endothelial cells (Krull et al.,
1997), delivery of heterologous antigens to the MHC class
I processing and presentation pathway (Darji et al.,
484 H. Repp et al.
1995a), induction of apoptosis (Guzman et al., 1996),
NF-kB activation (Kayal et al., 1999), and induction of
mucus exocytosis in intestinal cells (Coconnier et al.,
1998). All of these different cellular responses are Ca2+dependent and can be modulated by changes of the intracellular Ca2+ level (Berridge et al., 1998).
To date, no characterization of the pores formed by LLO
in the plasma membrane of viable host cells has been
reported. Attempts to detect LLO-formed pores in artificial
lipid bilayers have so far proved difficult (Goldfine et al.,
1995). In the present study, we used the patch-clamp
technique to detect and characterize the pore-forming
effects and the functional consequences of pore formation by L. monocytogenes and purified LLO in nucleated
host cells. We found that exposure of HEK293 cells to L.
monocytogenes as well as low concentrations of LLO
rapidly leads to the generation of plasma membrane
pores. Pore openings and closings are accompanied by
oscillations of the intracellular Ca2+ concentration that
result from a direct Ca2+ influx via the LLO-formed pores.
A Ca2+-dependent effect that can be observed immediately after pore opening is the activation of Ca2+dependent K+ channels. The finding that LLO induces
long-lasting Ca2+ oscillations may provide a mutual molecular basis for the broad spectrum of Ca2+-dependent signalling events and cellular effects that are mediated by
this toxin.
Results
Exposure of HEK293 cells to L. monocytogenes leads
to pore formation
The pore-forming effects of L. monocytogenes were
studied in HEK293 cells using the whole-cell configuration of the patch-clamp technique. This cell type is
particularly suitable as it is electrophysiologically well
characterized and possesses only low endogenous ion
channel activity. HEK293 cells exhibit a transient Ca2+
current that is activated upon depolarization to voltages
positive of -40 mV (Berjukow et al., 1996) and a voltagedependent, Ca2+-independent K+ current of small amplitude that is activated at membrane potentials positive of
-30 mV. To avoid activation of these endogenous ion
channels, the effects of L. monocytogenes were studied
at -50 mV or more negative membrane potentials. Figure
1A shows a typical recording obtained after exposure
of cells to wild-type L. monocytogenes. Starting at 270 ±
49 s (n = 12) after the application of the bacteria, pore
formation in the plasma membrane occurred that subsequently led to an increase in the membrane current
amplitude in the range of 0.5–2 nA (n = 12) at -50 mV
membrane holding potential. The recording in Fig. 1A
demonstrates clearly that the pores did not stay continuously open but oscillated between open and closed
states. Whereas the pore openings always exhibited
Fig. 1. Pore formation after exposure to
wild-type L. monocytogenes (L. m.).
A. Section from a 15 min recording. The trace
goes downwards after a pore opening and
upwards during a pore closing. When the
recording was stopped, the membrane current
amplitude was ~ -0.6 nA.
B. Pore formation by LLO (100 ng ml-1). The
inset shows the beginning of pore formation
on an expanded time and current scale. The
dotted lines indicate membrane current levels
corresponding to pore openings. The
membrane holding potential was -50 mV.
Note the different time and current scales in A
and B.
© 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 483–491
LLO-formed pores cause Ca 2+ oscillations
485
rapid kinetics, pore closings occurred either slowly or
abruptly.
LLO is responsible for the pore formation
by L. monocytogenes
When HEK293 cells were exposed to a Dhly mutant of L.
monocytogenes that lacks LLO expression (Guzman et
al., 1995), no pore formation was observed (n = 4). This
finding suggested that the generation of pores after infection with L. monocytogenes is exclusively dependent on
the presence of LLO, which was confirmed in further
experiments. Figure 1B shows a recording of the membrane current of a cell after application of purified LLO
(100 ng ml-1) at -50 mV membrane holding potential. In
comparison with the effect observed with bacteria, the first
pore appeared after a much shorter time period (10 ±
3 s; n = 4) after LLO application. The pore formation progressed rapidly, and in less than a minute the membrane
current amplitude, which depends on the pore sizes and
the amount of simultaneously open pores, reached a
value of more than -1.5 nA at -50 mV membrane holding
potential. The inset in Fig. 1B shows the beginning of the
pore formation by LLO at a higher resolution. Different
current levels can be clearly resolved.
LLO-formed pores have current amplitudes that are
multiples of a single channel event and exhibit low ion
selectivity
An amplitude histogram of the differences between successive membrane current levels after induction of pore
formation by L. monocytogenes is shown in Fig. 2A. Three
elementary pore current amplitudes of -45.9 ± 8.4 pA
(‘small pore’; n = 84; SD), -83.6 ± 10.9 pA (‘medium pore’;
n = 71; SD), and -125.7 ± 9.5 pA (‘large pore’; n = 30;
SD) at -50 mV membrane holding potential were identified by statistical analysis. The elementary currents of
the medium and the large pore are two- and threefold
multiples of the elementary current of the small pore. The
small and medium pores occur with almost the same
frequency whereas the large pore occurs less frequently
(Fig. 2A).
The current-voltage relationships of the LLO-formed
pores in HEK293 cells (n = 12) are shown in Fig. 2B.
Slope conductances of 1151 ± 64 pS for the small pore,
2074 ± 191 pS for the medium pore, and 2957 ± 256 pS
for the large pore at negative potentials were obtained.
Linear extrapolation to positive potentials resulted in
reversal potentials of -5 mV for the small pore, -6 mV for
the medium pore, and -4 mV for the large pore, which are
not significantly different from zero. Because physiological extra- and intracellular solutions were used, a rever-
© 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 483–491
Fig. 2. A. Amplitude histogram of the differences between
successive membrane current levels after the start of pore
formation induced by L. monocytogenes at -50 mV membrane
holding potential. The recordings of 12 cells were analysed (n =
191 events). Due to the small number (6) of pore opening events
with amplitudes larger than -160 pA, these data were excluded and
a sum of three Rosin–Rammler–Sperling–Weibull (RRSW)
functions was adjusted to the remaining 185 data points. Mean
elementary current amplitudes of -45.9, -83.6, and -125.7 pA were
calculated. The RRSW distributions were converted into the
corresponding probability density functions represented by the solid
line.
B. Current–voltage relationships of the small (), medium () and
large pore () measured after exposure to L. monocytogenes (n =
12). Linear regression analysis yielded slope conductances of
1151, 2074 and 2957 pS respectively. Error bars indicate SD.
sal potential close to zero indicates that the LLO-formed
pores are non-selective, at least for cations such as Na+
and K+.
Pore formation by LLO is concentration dependent
and transient
The extent of pore formation by LLO in the plasma membrane of HEK293 cells is dependent on LLO concentration. Figure 3 shows the progress of pore formation during
a 70 min recording period after application of LLO at a
concentration of 5 ng ml-1. In comparison with a 20-fold
higher concentration (see Fig. 1B), the appearance of the
first pore was greatly delayed. Furthermore, small pores
486 H. Repp et al.
Fig. 3. Long-term recording (~70 min) of pore
formation in a single cell after application of
LLO (5 ng ml-1). The membrane holding
potential was -50 mV.
predominated whereas medium and large pores were
seldom observed, which shows that the distribution of
pore size is dependent on LLO concentration. The amplitudes of the membrane current were much lower and did
not exceed a value of about -200 pA at -50 mV membrane holding potential (n = 3), indicating that the maximal
number of pores that were simultaneously open at a concentration of 5 ng ml-1 LLO was less than 5 per cell. About
1 h after the application of LLO, the frequency of pore
openings decreased to almost zero. This finding strongly
suggests that LLO molecules remain only transiently in
the plasma membrane.
Fig. 4D, fluorescence images are presented of the cell
whose Ca2+ trace is shown in Fig. 4A, which unambiguously demonstrates oscillations in intracellular Ca2+
concentration. Application of purified LLO (50 ng ml-1)
replicated the effects observed after exposure to bacteria, and Ca2+ oscillations were continuously present during
a recording period of 1 h (data not shown). In contrast,
when Ca2+ was replaced by Ba2+ in the extracellular solution, no Ca2+ oscillations occurred after exposure to LLO.
This indicates that the Ca2+ oscillations are caused by an
influx of extracellular Ca2+.
LLO-formed pores lead to intracellular Ca 2+ oscillations
Ca2+ influx occurs through LLO-formed pores and leads
to activation of Ca 2+-dependent K + channels
To determine whether the generation of LLO-formed
pores leads to changes in the intracellular Ca2+ level, we
monitored cells loaded with the fluorescent Ca2+ indicator
indo 1-AM. A few minutes after application of L. monocytogenes, the intracellular Ca2+ concentration increased
and showed oscillations during further monitoring (Fig.
4A–D). Two types of Ca2+ elevations were observed: in
some cells, sharp Ca2+ spikes with a return to control
levels between the spikes occurred predominantly (Fig.
4A), whereas prolonged Ca2+ peaks with wave-like
increases and decreases were recorded in other cells
(Fig. 4B and C). The kinetics and occurrence of these two
types of elevations varied between individual cells. We
also visualized the temporal changes in intracellular Ca2+
concentration using time-lapse confocal microscopy. In
To determine whether Ca2+ influx occurs through LLOformed pores or endogenous Ca2+ channels, we wished
to monitor a functional activity resulting directly from LLObased pore formation and Ca2+ influx. An immediately
measurable, functional consequence of an intracellular
Ca2+ increase is the activation of Ca2+-dependent K+ channels. Because native HEK293 cells do not exhibit such K+
channels, we investigated HEK293 cells that were transfected with the Ca2+-dependent K+ channel type hSK4
(Joiner et al., 1997). Figure 5A shows a typical example
of the activation of hSK4 channels after exposure to L.
monocytogenes. As shown in the inset of Fig. 5A, simultaneous monitoring of both pore formation by LLO and K+
channel activity showed that the activation started directly
after the initial pore opening. These combined events
© 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 483–491
LLO-formed pores cause Ca 2+ oscillations
487
Fig. 4. A–C. Oscillations of the intracellular
Ca2+ concentration induced by LLO-formed
pores. Cells loaded with indo 1-AM were
exposed to L. monocytogenes at time zero.
The traces represent examples of time
courses of the intracellular Ca2+ levels of three
different cells. E(a) represents the emission at
405 nm and E(b) emission at 460 nm.
D. Single frames from an image series of the
intracellular Ca2+ concentration. Ca2+
elevations are represented by a colour shift
from green to red. Originally, greyscale
images were captured every 2 s and then
converted into pseudo-colour images, as
shown. The trace presented in (A) was
recorded from the cell that is marked by white
arrows in (D). Arrows 1–4 below the trace in
(A) indicate the times when the images were
recorded.
occurred at a membrane holding potential of -50 mV.
Because the Ca2+ channels that have been so far
detected in HEK293 cells are not activated at this negative membrane potential (Berjukow et al., 1996), this
finding strongly suggested that the influx of extracellular
Ca2+ does not occur via Ca2+ channels but directly through
the LLO-formed pores. This was confirmed in further
experiments. When Cd2+ (1 mM), a blocker of all types of
voltage-gated Ca2+ channels (Randall, 1998), and SK&F
96365 (25 mM), a blocker of receptor-operated Ca2+
channels (Rink, 1990), were present in the extracellular
solution, hSK4 channels were still activated directly after
the start of the pore formation by LLO (n = 4; data not
shown).
The hSK4 channel activation was transient and led to
a large hyperpolarization of the membrane potential to a
value of -86 ± 1 mV (n = 12). In the experiment depicted
in Fig. 5A, the first K+ current peak was followed by
two further K+ current activations, indicating separate
increases of the intracellular Ca2+ level that also started
directly after pore openings. A non-selective membrane
current overlapped with the third K+ current activation and
was a result of progressive formation of further LLO
pores. In other cells, one to four transient K+ current
© 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 483–491
activations were observed before the non-selective membrane current became apparent. Figure 5B shows that
when extracellular Ca2+ was replaced by Ba2+ (n = 3), LLO
still led to pore formation but activation of hSK4 channels
did not occur, demonstrating that an influx of extracellular Ca2+ is required for the activation.
Discussion
LLO is a major virulence factor of the facultative intracellular Gram-positive pathogen L. monocytogenes (Portnoy
et al., 1988; Cossart et al., 1989). Despite being classified as a pore-forming haemolysin for more than 30 years
(Portnoy et al., 1992; Alouf, 2001), LLO has not yet been
directly demonstrated to cause pore formation in cells,
and pore formation has been largely deduced from the
cytolytic effects of LLO that require high toxin concentrations. Accordingly, an LLO concentration of at least 1 mg
ml-1 was needed to visualize oligomeric pores in lysed
erythrocyte membranes by electron microscopy (Jacobs
et al., 1998). However, there is increasing appreciation
that the effects on cellular signalling that are induced by
LLO occur at sublytic toxin concentrations (Darji et al.,
1995a; Guzman et al., 1996; Nishibori et al., 1996;
488 H. Repp et al.
Fig. 5. A. Effect of L. monocytogenes (L. m.)
on the whole-cell membrane current of a
HEK293 cell expressing the Ca2+-dependent
K+ channel type hSK4. The membrane
holding potential was -50 mV. Upward
movements in the recording trace are caused
by K+ efflux through hSK4 channels, and
downward movements are due to pore
openings. The inset shows the beginning of
pore formation on an expanded time scale
representing a period of 8 s. Note that no
LLO-formed pore is open after the complete
decrease of the first K+ current peak and that
the next pore opening is directly followed by a
further K+ current peak. During the third
K+ current activation, progressive pore
openings become visible as straight
downward movements in the recording trace.
B. Same conditions as in A but using a
Ca2+-free extracellular solution that contained
2 mM Ba2+.
Sibelius et al., 1996; Tang et al., 1996; Krull et al., 1997;
Coconnier et al., 1998; Kayal et al., 1999).
The present work directly demonstrates pore formation
by LLO in viable host cells and identifies intracellular Ca2+
oscillations as a functional consequence of LLOformed pores. We found that LLO at a concentration of
100 ng ml-1 within seconds led to the start of extensive
pore formation in the plasma membrane of HEK293
cells. A concentration of 5 ng ml-1 produced only a small
response that started after several minutes. These data
translate into a steep concentration-response relationship
with an estimated Hill slope of about 2.5, which implies
that at least three LLO molecules are required to create
a functional pore. It is likely that LLO-formed pores can
form clusters exhibiting synchronized opening and closing
states, as the observed conductances of the medium
and large pore are two- and threefold multiples of that
of the small pore. This hypothesis is supported by the
observation that multilevel conductance states result
from simultaneous open/shut events of clusters of two
or more identical channel units as in the case of Clchannels (Larsen et al., 1996), ion channels formed by
syringomycin E of Pseudomonas syringae pv. Syringae
(Kaulin et al., 1998), and Ca2+-permeable, non-selective
cation pores formed by polycystin-2 (Gonzalez-Perret et
al., 2001). The LLO-formed pores exhibit a linear current-
voltage relationship and a reversal potential close to zero,
indicating a non-selective permeability for at least monovalent cations.
Pore formation by LLO leads to long-lasting oscillations
of the intracellular Ca2+ concentration. Exposure of cells
to purified LLO induces the same effect as exposure to
native bacteria. This shows that L. monocytogenes can
use LLO as a kind of remote control to manipulate the
intracellular Ca2+ level without direct interaction with the
host cell. Ca2+ oscillations occur in all cells, persist for
at least one hour, and depend on the presence of extracellular Ca2+, with kinetics and time-courses that vary
between individual cells. If LLO molecules simply
punched blunt holes into the plasma membrane, one
would expect instead of Ca2+ oscillations a continuous
increase in the intracellular Ca2+ level and a breakdown
of cellular ion homeostasis. At the low LLO concentrations
used in this study, this is not the case. Thus, the oscillating Ca2+ increases most probably reflect a balance
between cellular mechanisms that maintain Ca2+ homeostasis and a pulsed influx of extracellular Ca2+ caused
by LLO-formed pores. It is not yet clear whether LLOdependent Ca2+ influx can also trigger Ca2+ release
from intracellular stores, which could contribute to the
observed Ca2+ oscillations.
Extracellular Ca2+ can enter the cell directly through
© 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 483–491
LLO-formed pores cause Ca 2+ oscillations
LLO-formed pores. Using the Ca2+-dependent K+ channel
type hSK4 as an indicator of an intracellular Ca2+
increase, we found that the activation of hSK4 channels
started directly after pore openings. This was also the
case under conditions where Ca2+ channels were completely blocked. This demonstrates that LLO-formed
pores allow the flow of extracellular Ca2+ into the cell independently of Ca2+ channels. Furthermore, LLO-formed
pores themselves exhibit ion channel-like properties.
Thus, they can apparently limit Ca2+ influx and contribute
to the prevention of Ca2+ overflow, because they are
not continuously open but oscillate between open and
closed states and persist only transiently in the plasma
membrane. The identification of LLO as a direct
manipulator of the intracellular Ca2+ level reveals a
novel mechanism of how Ca2+ elevations can
be induced. In J774 macrophage-like cells, Ca2+ elevations that were dependent on the presence of both
LLO and phosphatidylinositol-specific phospholipase C
were observed after exposure to L. monocytogenes
(Wadsworth and Goldfine, 1999). In these cells, Ca2+ entry
is mediated by receptor-operated Ca2+ channels, which
was shown using the Ca2+ channel inhibitor SK&F 96365
(Wadsworth and Goldfine, 1999). In renal epithelial cells,
a-haemolysin of uropathogenic E. coli induces Ca2+
oscillations, subsequently stimulating the release of the
cytokines IL-6 and IL-8 (Uhlen et al., 2000). In this case,
a-haemolysin activates both voltage-operated L-type
Ca2+ channels and inositol trisphosphate receptors of the
affected cells (Uhlen et al., 2000).
In general, Ca2+ signals modulate intracellular and intercellular signalling (Berridge et al., 1998), and oscillation
frequency can contribute to the specificity of gene expression (Dolmetsch et al., 1997; 1998). Thus, the observation that LLO causes Ca2+ oscillations provides a potential
molecular basis for the plethora of Ca2+-dependent signalling events and cellular effects that are mediated by
LLO during Listeria infection (Darji et al., 1995a; Guzman
et al., 1996; Nishibori et al., 1996; Sibelius et al., 1996;
Tang et al., 1996; Krull et al., 1997; Coconnier et al., 1998;
Kayal et al., 1999). The finding that the LLO-formed pore
itself directly manipulates the intracellular Ca2+ level of the
target cell reveals a new facet of bacterial toxins whereby
an otherwise highly pleiotropic cellular signal is effectively
harnessed to manipulate cellular functions at sites that
are distal to the bacterium.
Experimental procedures
Cell culture and transfections
HEK293 cells were maintained in a humidified, 6% CO2 atmosphere in a mixture of DMEM and Ham’s F12 medium (1:1, v/v)
containing 10% (v/v) fetal calf serum (PAN Systems, Aidenbach,
Germany) and 2 mM L-glutamine without antibiotics. Human
© 2002 Blackwell Science Ltd, Cellular Microbiology, 4, 483–491
489
Ca2+-activated K+ channel type hSK4 (in pcDNA3) was a gift from
Dr W. J. Joiner (Yale University School of Medicine, New Haven,
CT, USA). Transfection with hSK4 of HEK293 cells (2 ¥ 105) was
performed using 7.5 ml of lipofectin (Life Technologies, Karlsruhe,
Germany) premixed with 2 mg of maxiprep DNA. Stable hSK4transfected cells were selected using geneticin (400 mg ml-1; Calbiochem-Novabiochem, Bad-Soden, Germany), and clonal lines
were examined for the amplitude of the Ca2+-dependent K+
current as an indicator of the expression level of hSK4. Clone A3
was used to investigate the effect of L. monocytogenes on Ca2+activated K+ channels.
Bacterial strains and culture
The bacterial strains used in this study include the wild-type
L. monocytogenes strain EGD-e 1/2a (Chakraborty et al., 2000)
and Dhly, a mutant lacking LLO activity (Guzman et al., 1995).
Bacterial strains were grown in brain heart infusion (BHI) medium
(37 g l-1) at 37°C. Stock cultures were stored at -80°C in
Luria-Bertani (LB) broth containing 40% glycerol. For electrophysiological experiments, 1–2 colonies of bacteria were grown
for 16 h in 10 ml BHI medium. On the following day, 200 ml of the
bacterial suspension were diluted in 10 ml BHI medium and
further cultured for 3 h. The optical density of the 3 h culture was
0.7–0.8 when measured at 650 nm. One ml of the 3 h culture
was centrifuged for 3 min at 3500 g and washed twice with
extracellular solution (see Solutions and drugs). The pellet was
resuspended in 1 ml of extracellular solution, and the resulting
suspension was used in the electrophysiological experiments.
Purification of LLO was performed as described (Darji et al.,
1995b).
Electrophysiological recording
HEK293 cells were plated in 35 mm dishes 48 h prior to the
experiments. Cells (about 4 ¥ 105 per 35 mm dish) were washed
with extracellular solution (see Solutions and drugs), and the
recordings were started 10–30 min after the washing procedure
using a bath volume of 2 ml. 100 ml of a washed suspension containing about 8 ¥ 107 bacteria (corresponding to a multiplicity of
infection of about 200) or 100 ml of LLO at concentrations of 5,
50 or 100 ng ml-1 were applied with a pipette directly into the bath
solution 2–5 min after a stable recording configuration had been
obtained. All measurements were performed at 20–22°C.
Recording pipettes were pulled from borosilicate glass capillaries (Hilgenberg, Malsfeld, Germany) with an outer diameter of
1.5 mm and a wall thickness of 0.3 mm. After fire polishing, they
had a resistance of 5–10 MW when filled with pipette solution.
Initial seal resistances were approximately 5–10 GW. Whole-cell
currents were recorded with an EPC-9 patch-clamp amplifier
(HEKA GmbH, Lambrecht, Germany) filtered at 30 Hz or 3 kHz
and digitized at 100 Hz or 10 kHz respectively. Membrane potentials were measured at zero current in the current-clamp mode
of the whole-cell recording configuration. Data acquisition and
off-line analysis were performed with a Macintosh computer
Quadra 840 AV with Pulse and Pulsefit software (HEKA GmbH,
Lambrecht, Germany). The data were corrected for the liquidjunction potential between the pipette and bath solutions, which
was -10 mV for the standard K + glutamate internal solution. Data
are expressed as means ± SEM unless stated otherwise.
490 H. Repp et al.
Intracellular Ca2+ measurements
For intracellular Ca2+ measurements, HEK293 cells were plated
on 35 mm plastic dishes that had been modified for fluorescence
measurements. A hole of 15 mm diameter was drilled into the
bottom of a 35 mm plastic dish and covered with a glass coverslip that was fixed with silicone rubber (GE Silicones, Bergen op
Zoom, the Netherlands). On the day of infection, cells were
loaded for 45 min at 37°C with the fluorescent Ca2+ indicator indo
1-AM (Calbiochem-Novabiochem, Bad-Soden, Germany) dissolved in bath solution at a concentration of 2 mM. The solution
contained 16 mM Pluronic F-127 (Molecular Probes, Eugene,
USA) for a better dispersion of indo 1-AM. After the loading time,
HEK293 cells were washed twice with bath solution and further
incubated for 20 min at 37°C. Intracellular Ca2+ measurements
were performed at room temperature. Images were captured and
analysed with the Bio-Rad MRC1024 confocal imaging system.
The excitation wavelength was 352 nm and the emission wavelength was 460 nm for low [Ca2+] and 405 nm for high [Ca2+]. Upon
binding of Ca2+, the fluorescence emission maximum of indo 1 is
shifted to lower wavelengths. The obtained greyscale images
were converted into pseudo-colour images with Scion Image
software (Scion Corporation, Frederick, USA).
Solutions and drugs
Cells were bathed in extracellular solution composed of 140 mM
NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose,
10 mM HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulphonic
acid), pH 7.4 adjusted with NaOH. In some experiments, an
extracellular solution that contained BaCl2 (2 mM) instead of
CaCl2 was used. Differences in osmolarity between extraand intracellular solutions were compensated for as described
(Repp et al., 1998). The recording pipette contained an intracellular solution composed of 140 mM K+ glutamate, 10 mM
NaCl, 2 mM MgCl2, 10 mM HEPES, pH 7.3 adjusted with KOH.
A free intracellular Ca2+ concentration ([Ca2+]i) of 100 nM was
obtained using 100 mM of the Ca2+-chelator BAPTA (1,2-bis[2aminophenoxy]ethane-N,N,N¢,N¢-tetraacetic acid) and a total
Ca2+ concentration of 30 mM, assuming an apparent dissociation
constant KD of 0.24 mM (pH 7.3) for the Ca2+-BAPTA complex. For
experiments with hSK4-transfected HEK293 cells, a Ca2+-free
intracellular solution that contained 100 mM BAPTA (‘low
buffered’) was used. Bath and pipette solutions were filtered
through 0.2 mm pore filters (Renner, Dannstadt, Germany).
Acknowledgements
We thank W. J. Joiner for the generous gift of the hSK4 expression vector, J. Behrendt and S. Kocks for their contribution to the
Ca2+ measurements, and C. Zibuschka for expert technical assistance. This work was supported by grants from the Deutsche
Forschungsgemeinschaft (RE 1046/1–1, SFB 535 TPA5 and
TPB6, and Graduiertenkolleg ‘Molekulare Biologie und Pharmakologie’).
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