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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 29, 2001 as doi:10.1096/fj.00-0671fje. |
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* Lehrstuhl für Biotechnologie, Theodor-Boveri Institut (Biozentrum) der Universität Würzburg, D-97074 Würzburg, Germany;
Institut für experimentelle und klinische Pharmakologie und Toxikologie, Universität Freiburg, D-79104 Freiburg, Germany; and
Interactions Bactéries Cellules, Institut Pasteur, F-75724 Paris cedex 15, France
2Correspondence: Lehrstuhl für Biotechnologie, Theodor-Boveri Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany. E-mail: roland.benz{at}mail.uni-wuerzburg.de
SPECIFIC AIMS
C2-II, the binding component of ADP-ribosylating Clostridium botulinum C2 toxin, is involved in C2-mediated intoxication of target cells and forms channels in lipid bilayers. Here we studied the mechanism by which chloroquine and related compounds inhibit the C2-II channel in vitro and C2 toxin-mediated intoxication of Vero cells in vivo. In particular, we investigated the sidedness of this interaction and the relation between 4-aminoquinolone structure and channel function.
PRINCIPAL FINDINGS
1. Evaluation of the stability constant of the 4-aminoquinolone
binding to C2-II
Activated C2-II but not nonactivated C2-II forms ion-permeable
channels in lipid bilayer membranes. 4-Aminoquinolones known as potent
antimalarial drugs interact with the C2-II channel, thereby blocking
the channel. The block was studied using titration experiments. These
measurements allow the calculation of the stability constants for
4-aminoquinolone binding to the channel. C2-II was reconstituted into
lipid bilayers, then small amounts of concentrated chloroquine
solutions were added to the aqueous phase on both sides of the
membrane. The membrane conductance decreased as a function of the
chloroquine concentration. The data of Fig. 1
and of similar experiments with the analogs 4-aminoquinaldine,
4-amodiaquin, primaquine, quinine, and quinidine were analyzed using
Lineweaver-Burke plots. A stability constant (K) of
22,200 ± 1200 1/M (half saturation constant
KS=45 ±2.0 µM) was calculated from
the data for the binding of chloroquine to the C2-II channel. Similar
analyses were performed with the chloroquine analogs. The affinity of
chloroquine binding to C2-II in 1 M KCl was highest, followed by
primaquine, 4-amodiaquin, quinidine, quinine, and 4-aminoquinaldine
(see Table 1
).
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2. Chloroquine binding to C2-II is ionic strength dependent
Chloroquine is twofold positively charged at neutral pH. Part of
the binding between this molecule and the C2-II channel might be caused
by an interaction between oppositely charged groups localized at
chloroquine and at C2-II. Such an interaction should be ionic strength
dependent. The results of titration experiments at 0.1 M KCl suggested
that the stability constant of chloroquine binding to C2-II was indeed
drastically increased. The strongest effect was observed for
chloroquine; its stability constant increased by a factor of 
13 as
compared to 1 M KCl. The ionic strength effect on primaquine,
4-amodiaquin, 4-aminoquinaldine, and quinine binding was less
pronounced (see Table 1
).
3. The C2-II channel inserts oriented into the artificial lipid
bilayer membranes
Titration experiments can be performed in a symmetrical way, i.e.,
chloroquine and related compounds were added to both sides of the
membrane. However, it is also possible to study binding with respect to
a possible asymmetry of the C2-II channel. For this, the channel must
be reconstituted into the membranes in a fully oriented way. To test
this possibility, we added C2-II to only one side of the membrane and
studied its voltage dependence. Starting with about -30 to -40 mV
applied to the cis side of the membrane (the same side to
which C2-II was added), the current through the channels decreased
exponentially; for the opposite potential at the cis side of
the membrane, the current was voltage independent. This suggests
asymmetric insertion of the C2-II channel into the membrane and that
the channels are almost completely closed for high negative voltages at
the cis side of the channel.
4. Evaluation of the stability constant of chloroquine binding to
C2-II under asymmetric conditions
To investigate whether the binding of chloroquine to C2-II was
dependent on the orientation of the channel, we performed titration
experiments where chloroquine and C2-II were both added to the
cis side of the membrane. The stability constant for
chloroquine binding to the cis side of C2-II channel was
approximately the same as when it was added to both sides of the
membrane and a K of 340,000 ± 18,000 1/M was obtained
(0.1 M KCl, 4 experiments). The addition of chloroquine to the
trans side of the membranes demonstrated a much weaker
affinity for the C2-II channel. In this case, K was
13,000 1/M in 0.1 M KCl and 600 1/M im 1 M KCl. This means that
the C2-II channel exhibits considerable asymmetry for the binding of
chloroquine and other 4-aminoquinolones.
5. Chloroquine-mediated block of C2 toxin activity in vivo
We studied the effect of chloroquine on C2-I intoxication of Vero
cells. Cells were preincubated with different concentrations of
chloroquine and some related compounds at maximum concentrations, which
did not induce a cytotoxic effect, then C2-I and C2-II were added to
the Vero cells. KS for 50%
chloroquine-mediated inhibition of C2 toxin action on Vero cells was
23 ± 7.6 µM (K=43,500 1/M). This half saturation
constant is similar to that obtained from lipid bilayer measurements at
0.1 M KCl. High C2 toxin inhibition was also observed for
4-aminoquinaldine, quinine, and 4-amodiaquin. For primaquine and
quinidine, no inhibition of C2 toxin action on Vero cells could be
detected within the range where no cytotoxic effects could be observed.
This means that their half saturation constants are probably
considerably below those of chloroquine. Chloroquine itself exhibits
the highest toxin inhibition (half saturation concentration 23 µM).
Apparent half saturation constants could also be calculated for the
related compounds, which were between 20 and 50 µM (data not shown)
CONCLUSIONS
1. Chloroquine and related compounds block the C2-II-induced
channel in lipid bilayer membranes
The results presented here clearly show that chloroquine and other
4-aminoquinolones efficiently block the channels formed by the binding
component C2-II of the C2 toxin of C. botulinum, which
suggests a chemical reaction between a binding site inside the channel
and the different compounds. Its stability constant increases with
decreasing ionic strength in the aqueous phase. This means that one or
both positively charged groups of chloroquine interact with a
negatively charged environment inside the C2-II channel. The C2-II
channel has a diameter of 1 nm derived from ionic strength
dependence of the single-channel conductance caused by negatively
charged groups. These are presumably the same ones that interact with
the 4-aminoquinolones. There is probably not enough space for hydrated
ions to slide between the channel wall and the bulky chloroquine bound
inside the 1 nm wide channel. We presented good evidence that the C2-II
channel inserts asymmetrically into the lipid bilayer membranes when it
is added to only one side of the membrane. This was suggested by the
asymmetric response of the channel to the applied membrane potential
and the asymmetric binding with respect to the addition of C2-II and of
chloroquine to the same side of the membrane. This arrangement is
probably the same as in vivo, where both chloroquine and the binding
component are present on the surface of the target cell.
2. Chloroquine and related compounds inhibit intoxication of Vero
cells by C2 toxin in vivo
Another interesting result of this study is the inhibition of C2
toxin-mediated intoxication of Vero cells by the 4-aminoquinolones in
vivo. The half saturation constants for this inhibition are about the
same as those derived from blockage of the C2-II channel by the same
compounds in vitro. This suggests that chloroquine also hinders toxin
transport across the cytoplasmic (endosomal) membrane of the target
cells and thus protects them against C2 toxin action. This means not
only that the formation of the channels by C2-II is an important
prerequisite for the import of the toxin component into the target
cell, but that channels have to be open since we have demonstrated that
the closure of existing channels also inhibits toxin transport or at
least C2-I binding on C2-II.
3. Implication of chloroquine binding to the channel formed by the
C2-II binding component
There exists some sequence homology between the binding component
of C2 toxin, that of C. perfringens iota toxin and the
anthrax protective antigen. The latter has been crystallized in its
monomeric and heptameric form. The heptameric form is probably the one
that binds to the target cell membrane and inserts a 14-stranded
ß-barrel into the membrane. If the structure of the C2-II oligomer is
similar to that of the heptamer of the anthrax protective antigen, then
the channel-forming complex of the C2 binding component is highly
asymmetric, since most hydrophilic material would be localized on one
side of the membrane, the cis side of lipid bilayer
membranes or the surface of the target cell, and only a very small part
of the 350 kDa oligomer would be localized in the target cell membrane.
Such an arrangement is indeed suggested by the asymmetric response of
the C2-II channel to voltages of opposite polarity. This also means
that the 14-stranded ß-barrel cylinder contains in the channel lumen
seven at least partially negatively charged glutamic acids. The
positively charged quaternary ammonium groups of the 4-aminoquinolines
interact with them, which leads to the block of the narrow channel by
the bulky quinolone group. These negative charges are probably better
accessible from the cell surface side of the channel, which may explain
the sidedness of chloroquine binding. The results of the titration
experiments suggest that some of the 4-aminoquinolones have a higher
affinity for the C2-II channel. The distance between the amino and the
quinolone group and a second positive charge nearby is typical for
them. Besides chloroquine itself, primaquine and 4-amodiaquin also
belong to this class of molecules with higher binding affinity.
4-Aminoquinaldine and quinine contain only one positive charge. These
compounds have a somewhat smaller affinity toward the C2-II channel.
Our results demonstrate that chloroquine and some of the related
compounds block intoxication by C2 toxin in vivo. A possible mechanism
for the 4-aminoquinolone-mediated inhibition of intoxification is shown
Fig. 2
. Chloroquine may either hinder the binding of C2-I to C2-II or block
its transport through the C2-II channel in the early endosome.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0671fje ; to cite this
article, use FASEB J. (May 29, 2001) 10.1096/fj.00-0671fje 
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