From Centre National de Référence
Anaérobies, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris,
cedex 15, France and § Lehrstuhl für Biotechnologie,
Theodor-Boveri-Institut (Biozentrum) der Universität
Würzburg, Am Hubland, D-97074 Würzburg, Germany
Received for publication, November 16, 2000, and in revised form, February 7, 2001
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ABSTRACT |
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Epsilon toxin is a potent toxin produced
by Clostridium perfringens types B and D, which are
responsible for a rapidly fatal enterotoxemia in animals. One of the
main properties of epsilon toxin is the production of edema. We have
previously found that epsilon toxin causes a rapid swelling of
Madin-Darby canine kidney cells and that the toxin does not
enter the cytosol and remains associated with the cell membrane by
forming a large complex (Petit, L., Gibert, M., Gillet, D.,
Laurent-Winter, C., Boquet, P., and Popoff, M. R. (1997) J. Bacteriol. 179, 6480-6487). Here, we report that epsilon toxin
induced in Madin-Darby canine kidney cells a rapid decrease of
intracellular K+, and an increase of Cl Epsilon toxin is synthesized by Clostridium perfringens
types B and D. This toxin is the main virulence factor of C. perfringens type D, which is responsible for enterotoxemia in
sheep, goat, and more rarely in cattle. Enterotoxemia is a rapidly
fatal disease that causes important economical losses through the world.
Overgrowth of C. perfringens type D in the intestine of
susceptible animals is accompanied by the release of large amounts of
epsilon toxin, which is absorbed through the intestinal mucosa and
diffuses to all the organs by the blood circulation. The main biological activity of epsilon toxin is the production of edema. In
experimental animals, epsilon toxin elevates the blood pressure, increases the vascular permeability, and causes edema and congestion in
various organs including lungs and kidneys. Necrosis of the kidneys
is also observed in lambs that have died from enterotoxemia (1, 2).
Epsilon toxin is able to cross the blood-brain barrier and accumulates
in the brain (3, 4). The terminal phase of enterotoxemia is
characterized by neurological disorders (opisthotonus, convulsions, and
agonal struggling). Epsilon toxin increases the permeability of the
brain vasculature and causes perivascular edema, which is probably
responsible for neuronal damage and neurological disorders (5, 6). In
addition, epsilon toxin could directly interact with hippocampus
neurons leading to an excessive release of glutamate (7, 8).
Epsilon toxin is secreted as a non-toxic precursor that is activated by
Epsilon toxin is cytotoxic for
MDCK1 cells and at a lesser
extent for the human leiomyoblastoma (G-402) cells (11-13). We have previously found that epsilon toxin induces swelling, blebbing, and
lysis of MDCK cells and that the toxin does not enter the cytosol and
remains associated with the MDCK cell membrane by forming a large
complex (14). The cytotoxic activity was correlated with the formation
of a large membrane complex and an efflux of K+ (14, 15).
In addition, epsilon toxin causes a rapid decrease of the
transepithelial resistance of MDCK cell
monolayers.2
Here, we report that in MDCK cells, epsilon toxin causes a rapid
decrease of intracellular K+, a rapid increase of
Cl Materials--
Epsilon toxin was purified as previously
described (14). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) and propidium iodide were from Sigma. The
fluorescent probes CD222, sodium green, calcium green, and MQAE were
from Molecular Probes.
Cell Culture--
MDCK cells were grown in DMEM supplemented
with 10% fetal calf serum at 37 °C in a 5% CO2
incubator. For cytotoxicity and propidium assays, MDCK cells were grown
to confluency in 96-well plates. The monolayers were washed once in
DMEM and incubated with serial dilutions of epsilon toxin in DMEM
(100-µl final volume in each well). The viability test using MTT was
performed as described previously (14).
Intracellular Ion Assays--
MDCK cells were grown in 6-well
plates in DMEM supplemented with 10% fetal calf serum until
confluence. After two washings with DMEM without serum, the cells were
incubated with epsilon toxin (10 Propidium Iodide Influx--
For assay of PI entry, MDCK cells
were grown on 96-well plates until confluency. PI (5 µg/ml) was added
in the culture medium, together with epsilon toxin. At the indicated
times, the plates were read with a spectrofluorimeter (Fluoroskan II;
excitation 540 nm and emission 620 nm). The results were expressed as
the percentage of fluorescence obtained by treatment with Triton X-100 (0.2%) for 30 min at 37 °C.
Lipid Bilayer Experiments--
Black lipid bilayer membranes
were formed as has been described previously (16) from a 1% solution
of diphytanoyl phosphatidylcholine, phosphatidylserine, and asolectin
(Avanti Polar Lipids, Alabaster, AL) in N-decane. The
instrumentation consisted of a Teflon chamber with two aqueous
compartments connected by a small circular hole with a surface area of
0.3 to 0.5 mm2. The lipid bilayer membranes were formed by
painting onto the holes solutions of the different lipids. The aqueous
salt solutions (Merck) were used unbuffered and had a pH value around
6, if not indicated otherwise. Native and activated C. perfringens epsilon toxin were added from concentrated stock
solutions to the aqueous phase bathing membranes in the black state.
The temperature was kept at 20 °C throughout. The single-channel
recordings were performed using a pair of Ag/AgCl electrodes with salt
bridges switched in series with a voltage source and a highly sensitive
current amplifier. The amplified signal was monitored with a storage
oscilloscope, and the reconstitution of channels in the black lipid
membrane was recorded with a strip chart recorder. Zero-current
membrane potential measurements were performed by establishing a salt
gradient across membranes containing 100 to 1000 epsilon toxin channels as they have been described earlier (17).
Epsilon Toxin Induces in MDCK Cells an Early Loss of
K+, an Entry of Na+ and Cl Epsilon Toxin Induced a Cell Permeability to Propidium
Iodide--
The pore-forming toxins such as Staphylococcus
aureus
To evaluate the size of the pores caused by epsilon toxin, competition
with uncharged compounds of various molecular mass were
performed to block the channels induced by epsilon toxin and
subsequently to inhibit the effects of the toxin on cell viability and
entry of PI into MDCK cells. Such techniques have been used previously
with various hemolysins, for example from Escherichia coli,
Clostridium septicum, or S. aureus.
(20-22). Glucose (300 mM), sucrose (300 mM),
and PEG300 (25 mM) impaired neither the cytotoxic activity
in the MTT test nor the entry of PI induced by epsilon toxin. PEG6000
but not PEG3350 (25 mM) blocked the entry of PI stimulated
by epsilon toxin (data not shown). However, these data were not highly
reliable, because PEG1000 (25 mM) and above were to some
extent cytotoxic for MDCK cells. In any case the results indicate that
epsilon toxin forms large pores in MDCK cells at least 2 nm in diameter
based on the size of PEG1000 (23).
Activation Increases the Channel-forming Activity of C. perfringens
Epsilon Toxin in Lipid Bilayer Membranes--
The in vivo
experiments indicated that epsilon toxin formed pores that were at
least permeable to ions. Thus, it could be possible that epsilon toxin
forms ion-permeable channels in membranes. In a first set of
experimental conditions we studied the effect of the non-activated
epsilon toxin on membranes formed from a variety of different lipids
such as phosphatidylcholine, phosphatidylserine, and asolectin. In all
cases, we were able to observe some low channel-forming activity, which
means that only a small number of channels were formed even at very
high concentrations of non-activated epsilon toxin. Substantially
increased channel-forming activity was observed, however, when epsilon
toxin was activated by trypsin treatment before its addition to the
aqueous phase bathing lipid bilayer membranes. Again, we did not
observe any lipid specificity. Approximately, the same membrane
activity was observed for all lipids used in this study. The relation
between membrane conductance and toxin concentration in the aqueous
phase was linear, which suggests that there is no
association-dissociation reaction between non-conducting monomers and
conducting oligomers and that the conductive units are long-lived.
Fig. 4 shows the results of a lipid
bilayer experiment with activated C. perfringens epsilon
toxin. After formation of a diphytanoyl phosphatidylcholine/N-decane membrane in a 1 M
KCl solution, we added 80 ng/ml of trypsin-activated epsilon toxin to
the same side of the membrane. After about 2 min the membrane
conductance started to increase in a stepwise fashion. The membrane
conductance increased subsequently by more than three orders of
magnitude within 30 min. Interestingly, the channels showed a stepwise
fashion similar to the reconstitution of Gram-negative bacterial porins into lipid bilayer membranes (24). This means that they were mostly in
the open configuration and did not close under our experimental conditions. This result indicated that the channel was formed by a
defined structure, which does not show an association-dissociation equilibrium, such as the oligomers that form the hemolysin channels of
Proteus vulgaris and Morganella morganii (25). A
histogram of the channels formed by the activated C. perfringens epsilon toxin under the conditions of Fig. 4 is shown
in Fig. 5. The epsilon toxin channel had
on average a single-channel conductance of 550 pS in 1 M
KCl. Fig. 5 demonstrates that the current fluctuations were very
homogeneous, because the single-channel conductance ranged under the
conditions of Fig. 4 from 440 to ~640 pS, and other conductance
values were only rarely observed. The most frequent single-channel
conductance was 560 pS.
Properties of the C. perfringens Epsilon Toxin
Channel--
Single-channel experiments were also performed with salts
other than KCl to obtain some information on the size and selectivity of the epsilon toxin channel. The results are summarized in Table I. The replacement of chloride and
potassium by the less mobile acetate and lithium ions had a
considerable influence on the single-channel conductance. The influence
of the replacement of chloride by acetate on the single-channel
conductance was, however, more substantial (see Table I), which
suggests that the channel formed by epsilon toxin in lipid bilayer
membranes is at least slightly anion-selective. Table I shows also the
average single-channel conductance, G, as a function of the KCl
concentration in the aqueous phase. We observed a 1:1 relationship
between conductance and KCl concentration, which would be expected for
wide, water-filled channels similar to those formed by general
diffusion porins of Gram-negative bacteria (24). This probably means
that the channel formed by the C. perfringens epsilon toxin
represents a general diffusion pore.
Selectivity of the Epsilon Toxin Channel--
The selectivity of
the epsilon toxin channel was measured in zero-current membrane
potential measurements in the presence of salt gradients. After
incorporation of a large number of channels in membranes bathed in 100 mM KCl, 5-fold salt gradients were established across the
membranes by the addition of small amounts of concentrated KCl solution
to one side of the membrane. In all cases, the more diluted side of the
membrane became negative, which indicated preferential movement of
anions through the epsilon toxin channel, i.e. it
is anion-selective as was already suggested from the single-channel
data (Table I). The zero-current membrane potential for a 5-fold KCl
gradient was on average about C. perfringens Epsilon Toxin Forms Pores Permeable to Ions and PI
in MDCK Cells--
As previously reported, the in vivo
experiments suggest that epsilon toxin forms a complex in the membranes
of the MDCK cells and does not enter their cytoplasm. The formation of
the complex of the activated toxin molecule results in the loss of ions
from the cells and in cell death (14, 15, 26). These results suggest
that C. perfringens epsilon toxin destroys the barrier function of the cytoplasmic membrane of MDCK cells but not of other
cells, which probably do not contain a cell surface receptor for the
toxin. Here, we show that epsilon toxin causes in MDCK cells a very
early loss of K+ and entry of Na+ and
Cl C. perfringens Epsilon Toxin Forms Channels in Lipid Bilayer
Membranes--
In agreement with the possible destruction of the
barrier function of the cytoplasmic membrane of MDCK cells, we were
able to identify channel formation in artificial lipid bilayer
membranes. Essential for the formation was the trypsin-mediated
activation of the protein. No obvious lipid specificity was detected in
the experiments. Channels were formed with all lipids tested here. This
result seems to represent a contradiction to receptor-mediated destruction of cells. However, it has to be kept in mind that many
cytolytic bacterial toxins, such as the repeats in toxin toxins
(28-30),
It is noteworthy that the formation of channels in lipid bilayers
mediated by C. perfringens epsilon toxin was not a rare event. The addition of 100 ng/ml of this protein to the aqueous phase
bathing a diphytanoyl phosphatidylcholine/N-decane membrane was able to increase the conductance of lipid bilayer membranes considerably, and more than 1000 channels were formed within about 20 to 30 min under these conditions in a membrane with a surface area of
about 0.4 mm2. Higher concentrations led to the formation
of even more channels. These considerations and the observation of
channel formation of epsilon toxin in vivo suggest that we
are not dealing with an unspecific artifact.
Properties of the C. perfringens Epsilon Toxin Channel in Lipid
Bilayer Membranes--
The epsilon toxin channel is presumably formed
by protein oligomers with an apparent molecular mass of about 155 kDa
when inserted into cell membrane (14). This makes about five times the
molecular mass of a monomer. It is noteworthy that a common architecture of cytolytic toxins is the heptamer, which has been found
for
Besides oligomer formation the epsilon toxin channel also shares some
other features with those formed by aerolysin and and
Na+, whereas the increase of Ca2+ occurred
later. The entry of propidium iodide that was correlated with the loss
of cell viability monitored by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test
indicates that epsilon toxin formed large pores. In artificial lipid
bilayers, epsilon toxin caused current steps with a single-channel
conductance of 60 pS in 100 mM KCl, which represented
general diffusion pores. The channels were slightly selective for
anions, but cations could also penetrate. Epsilon toxin formed wide and
water-filled channels permeable to hydrophilic solutes up to a
molecular mass of at least 1 kDa, which probably represents the basic
mechanism of toxin action on target cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
protease produced by C. perfringens or by digestive proteases such as trypsin and
-chymotrypsin. The proteolytic activation is achieved by removal of 11-13 N-terminal and 22-29 C-terminal amino acids depending of the protease (9). For example, trypsin releases 13 N-terminal and 22 C-terminal amino acids, and
C. perfringens
protease removes 11 N-terminal and 29 C-terminal amino acids (9). This induces a significant change of the
isoelectric points (8.3 for the prototoxin and 5.4 for the activated
toxin) (10).
and Na+, and a slower increase of
Ca2+. The entry of propidium iodide (PI) into treated cells
indicates that epsilon toxin forms large membrane pores.
Investigation with artificial lipid bilayers shows that epsilon toxin
elicits the formation of non-selective general diffusion channels
permeable to molecules up to 1 kDa.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8 M) in DMEM
without serum (0.5 ml per well) at 37 °C in a CO2 incubator. At the indicated times, the cells were washed with 25 mM MOPS (pH 7) containing 150 mM glucose
(MOPS-glucose) and lysed with 1% Triton X-100 (125 µl per well).
Each fluorescent probe was added to 45 µl of lysed cells to a final
concentration of 1 µM for CD222 (K+ assay) or
10 µM for sodium green, calcium green, and MQAE
(Cl
assay). The probes were measured in a
spectrofluorimeter (Fluoroskan II; Labsystems) with the following
filters: excitation 380 nm and emission 475 nm for CD222, excitation
485 nm and emission 538 nm for sodium green and calcium green, and
excitation 355 nm and emission 475 nm for MQAE. The fluorescence
obtained by washing MDCK cells with MOPS-glucose and lysing with 1%
(w/v) Triton X-100 addition was considered as the 0% base line, and the fluorescence of MDCK cells incubated with serum-free DMEM containing 0.2% (w/v) Triton X-100 for 5 min at 37 °C in the
absence of epsilon toxin and then washed with MOPS-glucose and lysed
with 1% (w/v) Triton X-100 was considered as an activity of 100%. The data were expressed as percents of fluorescence quench.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and a
Later Influx of Ca2+--
We have previously reported that
epsilon toxin causes a rapid swelling of MDCK cells and a subsequent
loss of viability as monitored by the MTT test (14). Because epsilon
toxin interacts with the cell membrane and has apparently no
intracellular activity, its mechanism of action could consist in pore
formation, as it is the case for many hemolysins. An increase of the
membrane permeability for K+ has been evidenced in MDCK
cells damaged by epsilon toxin (14, 15). To define more precisely the
membrane permeability changes during the intoxication process of
epsilon toxin, we analyzed the kinetics of intracellular cations
(K+, Na+, and Ca2+) and anion
(Cl
) versus the cytotoxic activity determined
by the MTT test. Fig. 1 shows that
intracellular K+ decreased rapidly. The 50% loss of
K+ was observed within the first 5 min, whereas the 50%
decrease of cell viability was recorded after 30 min of incubation with 10
8 M of epsilon toxin. At this time, more
than 90% of intracellular K+ was lost. The intracellular
concentrations of the other cations, Na+ and
Ca2+, which are low in control cells, increased
significantly in MDCK cells intoxicated with epsilon toxin (Figs. 1 and
2). The Na+ entry was rapid
(50% increase of intracellular Na+ during 8 min) although
slightly later than the loss of K+, but that of the
divalent cation Ca2+ was delayed (50% increase of
intracellular Ca2+ during 26 min). The increase of the
intracellular Cl
concentration was as rapid as the
decrease of K+ (50% decrease during 5 min). This
demonstrates that the intracellular concentrations of K+,
Na+, Ca2+, and Cl
changed
dramatically in MDCK cells treated with epsilon toxin. The early
intracellular concentrations changes concerned that of the monovalent
ions K+, and Cl
and slightly delayed for
Na+, whereas that of the divalent cation Ca2+
was slower.
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Fig. 1.
Intracellular concentrations of
K+ and Na+ during the intoxication process of
MDCK cells with epsilon toxin. MDCK cells were grown on 6-well
plates and were treated with epsilon toxin (10 8
M) for the indicated times. Cell viability was tested with
the MTT test (X), and intracellular concentrations of
K+ (
) and Na+ (
) were measured by
spectrofluorimetry. Data are means ± S.D. (n = 6).
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Fig. 2.
Intracellular concentrations of
Cl and Ca2+ during the intoxication process
of MDCK cells with epsilon toxin. MDCK cells were grown on 6-well
plates and were treated with epsilon toxin (10
8
M) for the indicated times. Intracellular concentrations of
Cl
(
) and Ca2+ (
) were measured by
spectrofluorimetry. The data of cell viability are the same as in Fig.
1. Data are means ± S.D. (n = 6).
-toxin recognizes a cell surface receptor,
oligomerizes, and inserts into the membrane (18). At high concentration
(>300 nM)
-toxin forms large pores permitting the entry
of PI into cells (19). PI internalization was assayed in MDCK cells
treated with epsilon toxin. Fig. 3 shows
that the entry of PI correlated with the loss of cell viability
measured by the MTT test. The 50% entry of PI matched the 50%
increase of intracellular Ca2+ (at 26-27 min), and both
are close to the 50% loss of cell viability (at 32 min) induced by
epsilon toxin intoxication. This indicates that epsilon toxin induced
the formation of large pores in MDCK cell membranes even at a low
concentration (10
8 M) similar to
-toxin.
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Fig. 3.
Cell viability and entry of propidium iodide
in MDCK cells treated with epsilon toxin. MDCK cells were grown on
96-well plates and were treated with epsilon toxin (10 8
M) for the indicated times. Propidium iodide (+) was
monitored by spectrofluorimetry. The data of cell viability are the
same as in Fig. 1. Data are means ± S.D. (n = 6).
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Fig. 4.
Single-channel recording of a diphytanoyl
phosphatidylcholine/N-decane membrane in the presence
of activated epsilon toxin from C. perfringens.
10 min after the formation of the membrane 80 ng/ml of activated
epsilon toxin was added to the aqueous phase on one side of the
membrane. The aqueous phase contained 1 M KCl (pH 6). The
applied membrane potential was 50 mV; T = 20 °C.
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Fig. 5.
Histogram of the probability of the
occurrence of certain conductivity units observed with membranes formed
of diphytanoyl phosphatidylcholine/N-decane in the
presence of 80 ng/ml of activated epsilon toxin from C. perfringens. The aqueous phase contained 1 M KCl. The applied membrane potential was 50 mV;
T = 20 °C. The average single-channel conductance
was 550 pS for 255 single-channel events. Single-channel recordings of
6 different membranes were analyzed.
Average single-channel conductance, G, of the channel formed by the
activated epsilon toxin of C. perfringens in different salt solutions
19 mV at the more diluted side (mean of
four experiments). Analysis of the zero-current membrane potentials
using the Goldman-Hodgkin-Katz equation (17) suggested that cations
could also have a certain permeability through the epsilon toxin
channel, because the ratio of the permeabilities PK and
PCl was 0.30 (± 0.02; see Table
II). This result represents another
indication that the C. perfringens epsilon toxin forms a
general diffusion pore, because both anions and cations (albeit at
smaller rate than the anions) could penetrate the channel, and their
ratios reflect their mobility sequence.
Comparison of the channel properties of C. perfringens epsilon-toxin
with those of aerolysin and of alpha-toxin
-toxin were taken from Ref. 31.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, whereas the intracellular concentration of the
divalent cation Ca2+ increased later similarly to the loss
of cell viability monitored by the MTT test. Thus, epsilon toxin seems
to form not very selective pores through the MDCK cell membrane
permitting the flux of different ions. Differences in fluxes of
monovalent and divalent cations have also been observed with other
pore-forming toxins. As an example, staphylococcal
-toxin at low
concentrations creates in susceptible cells such as keratinocytes pores
permeable to K+ but not to Ca2+, and at high
concentrations a rapid flux of K+ and Ca2+ is
observed. The heterogeneity of pore sizes remains unexplained (27). The
pores induced by epsilon toxin were also permeable to PI. Inhibition of
PI entry with PEG of different molecular mass indicated that the pores
were large in size (at least 2 nm in diameter), but this is tentative,
because high molecular mass PEG were cytotoxic for MDCK cells. The cell
entry of PI induced by epsilon toxin matched the loss of cell
viability. A good correlation was observed between the kinetics of PI
entry and that of the MTT test. This raises the question whether the
pores formed by epsilon toxin in MDCK cells are responsible alone for
the loss of viability by release of ions and other essential molecules in the external medium. Thus, we cannot exclude the possibility that
epsilon toxin may elicit additional cellular activities such as to
trigger an intracellular signaling leading to cell death.
-toxin from S. aureus (31-33), and aerolysin
from Aeromonas sobria (31, 34) form channels in lipid
bilayer membranes without the need of receptors, whereas they all need
a receptor for biological activity. Lipid bilayers have smooth surfaces
without any surface structure including the surface-exposed
carbohydrates of biological membranes, which means that the toxins can
interact with the hydrocarbon core of the lipid bilayer and can insert without the help of receptors, although receptors may promote such an
interaction (35).
-toxin from S. aureus (33), aerolysin from
Aeromonas hydrophila (34), and the cytolytic toxin ClyA of
E. coli (36, 37). If the epsilon toxin oligomer has a
somewhat different mobility in SDS polyacrylamide gel electrophoresis
than the monomer, it is also possible that the membrane channel is
formed by a heptamer of the 30-kDa monomer. In such a case the
membrane-spanning part of the epsilon toxin should be formed by
-strands, as it is the case at least for
-toxin and aerolysin
(33, 34). In fact, secondary structure prediction performed with the
primary structure of epsilon toxin (10) reveals several stretches in
the protein that can possibly form membrane-spanning amphipathic
-strands.
-toxin. All three
channels are long-lasting channels with lifetimes in the range of
minutes. They are all anion-selective channels caused probably by an
excess of positively charged groups in or near the channel (see Ref. 31
and Table II). Nevertheless, all three toxin channels represent general
diffusion pores, because they do not contain selectivity filters or
binding sites for ions, as judged from the linear dependence of the
single-channel conductance from the aqueous salt concentration observed
for all three channels. This probably also means that the channels are
wide and water-filled and are permeable to solutes up to a molecular
mass of at least 1 kDa. Furthermore, the single-channel conductance
itself is also very similar in all three systems as Table II clearly
indicates. We did not find any similarity between the sequences of the
three toxins. However, this does not represent a serious problem for our assumption, because aerolysin and
-toxin do also not show sequence similarities despite a similar architecture of the membrane channel.
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FOOTNOTES |
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* This study was supported in part by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 487, Project A5) and the Fonds der Chemischen Industrie and by a Caisse Autonome Nationale d'Assurance Maladíe fellowship (to L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 33 1 45 68 83 07; Fax: 33 1 40 61 31 23; E-mail: mpopoff@pasteur.fr.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M010412200
2 Manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: MDCK, Madin-Darby canine kidney; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide; PI, propidium iodide; MOPS, 4-morpholinepropanesulfonic acid; PEG, polyethylene glycol.
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