(Received for publication, April 25, 1995; and in revised form, June 1, 1995)
From the
Protective antigen (PA), a component of anthrax toxin, mediates
translocation of the toxin's lethal and edema factors (LF and EF,
respectively) to the cytoplasm, via a pathway involving their release
from an acidic intracellular compartment. PA
Many toxic proteins are multifunctional enzymes that are able to
enter cells and covalently modify intracellular substrates. After
binding to a cell-surface receptor, these toxins generally undergo
receptor-mediated endocytosis and are transported to an appropriate
intracellular compartment, where their enzymic moieties are transferred
across the membrane and into the cytosol. Such toxins are usually
composed of two structurally distinct moieties, termed A and B, with B
serving to bind the toxin to receptors and facilitate transfer of the
enzymically active A moiety to
cytoplasm(1, 2, 3) . The precise mechanism by
which the B moiety promotes membrane traversal by A is yet to be
established in detail for any toxin. Bacillus anthracis,
the causative agent of anthrax, secretes three proteins: edema factor
(EF, ( According to
our current concept of anthrax toxin action(4) , the entry of
EF or LF into cells proceeds as follows. PA binds to a specific cell
surface receptor(9) , and its NH PA is capable
of forming ion-conductive channels in artificial bilayers and cellular
membranes(14, 15, 16) , and in both systems
proteolytic activation of full-length PA and exposure of the PA Recently, we found that purified PA If the PA
The percentage of released
Permeabilization of the plasma membrane of CHO-K1 cells by PA
was monitored by measuring
Figure 1:
Inhibition of PA-mediated
The inhibition of
Figure 2:
Concentration dependence of the inhibition
of
Figure 3:
pH dependence of the inhibition of
Because channel
formation by nicked PA alone shows a strong pH dependence (Fig. 3)(16) , we performed an experiment to
differentiate the pH dependence of the inhibition by LF from that of
channel formation.
The inhibition of
Figure 4:
Effect of LF on PA
To probe the possibility that LF might
nonetheless have an effect on the initial rate of oligomerization, we
performed an experiment in which the rate of oligomer formation was
monitored at pH 4.6 and 5.0 in the presence and absence of LF. As shown
in Fig. 5, oligomerization was essentially complete by 5 min at
pH 4.6, and inclusion of LF in the acidification buffer had little or
no effect. At pH 5.0, however, oligomer formation proceeded more
slowly, and a slight, but reproducible, inhibition by LF was seen.
Figure 5:
Kinetics of low pH-induced PA
Thus, while the inhibition of channel formation by LF might account
for part of the inhibition of
Figure 6:
Effect of LF on PA-mediated influx of
The ability of LF to inhibit PA The fact that LF and LF Half-maximal inhibition of
the PA ion channels in our experiments occurred at approximately 40
nM LF at pH 5.0 (Fig. 2). An apparent dissociation
constant of 10 pM of LF for PA We have previously established a relationship between
PA The role that either the oligomer or the channel plays in transfer
of EF and LF across the endosomal membrane into the cytosol is not yet
understood, but at least two models can be proposed. In the first, EF
and LF are assumed to traverse the endosomal membrane via the PA The block in channel conductance caused by LF at
acidic pH apparently results from the binding of LF to a site on
PA The proposed models for PA membrane insertion and LF/EF
translocation are likely to be directly relevant to translocation by
binary toxins produced by other bacteria, and perhaps to translocation
by structurally unrelated toxins(24, 25) . Diphtheria,
tetanus, and botulinum toxins share with anthrax toxin the ability to
insert into lipid bilayers and form ion channels under acidic
conditions(26) . In planar phospholipid bilayers the B moieties
of these binary toxins form voltage-dependent channels under acidic pH
conditions (27, 28, 29) . Understanding of
how the B moieties of the binary toxins facilitate transfer of their
respective A moieties across membranes and how ion channels participate
in this process, may reveal common features in the translocation
process. Addendum- After this work was completed, we
obtained sufficient quantities of recombinant EF to demonstrate that
this protein causes an inhibition of PA
, a 63-kDa
proteolytic fragment of PA, can be induced to form ion-conductive
channels in the plasma membrane of mammalian cells by acidification of
the medium. These channels are believed to be comprised of dodecyl
sulfate-resistant oligomers (heptameric rings) of PA
seen
by electron microscopy of the purified protein. Here we report that the
PA
-mediated efflux of
Rb
from preloaded CHO-K1 cells under acidic conditions is strongly
inhibited (
70%) by LF or LF
, a PA-binding fragment of
LF. Control proteins caused no inhibition. Evidence is presented that
the inhibition involves partial blockage of ion conductance by the
PA
channel. Also, oligomer formation is slowed somewhat by
LF at pH values near the pH threshold of channel formation (pH
5.3), suggesting that channel formation may also be retarded under
these conditions. The relevance of these results to the location of the
LF-binding site on PA
and the mechanism of LF and EF
translocation is discussed.
)89 kDa), lethal factor (LF, 90 kDa), and protective
antigen (PA, 83 kDa), which have been collectively termed anthrax
toxin. EF and LF serve as alternative A moieties, which act in
combination with PA, the B moiety, to generate different toxic effects
on cells(4) . EF is a calcium/calmodulin-dependent adenylate
cyclase, whose entry into the cytosol elevates the level of
cAMP(5) , causing various effects, depending on the nature of
the cell. In contrast, LF only affects macrophages, causing them to
produce high levels of interleukin 1 and tumor necrosis factor
(6) , which in turn induce shock and death of the host.
High concentrations of LF+PA result in cytolysis of
macrophages(7) . Although the biochemical activity of LF has
not yet been elucidated, analysis of its primary structure suggests
that the protein may be a metalloprotease(8) .
-terminal 20-kDa
domain (PA
) is removed by a cellular protease, implicated
as furin(10) , leaving the COOH-terminal 63-kDa domain
(PA
) bound to the receptor(11) . Removal of
PA
exposes a site on PA
to which either EF or
LF can bind(4) . The complex of receptor-bound
PA
EF or PA
LF then undergoes
receptor-mediated endocytosis and is delivered to an acidic
membrane-bound compartment, presumably the endosome. There, PA
facilitates translocation of EF and LF across the endosomal
membrane into the cytosol(12, 13) .
fragment to acidic pH are required for membrane insertion and
channel formation. The relevance of proteolytic activation and exposure
to acidic pH has been demonstrated in vivo: mutation or
deletion of the protease cleavage site in PA abolishes EF and LF
binding(10, 17) , and lysosomotropic agents protect
cells from the PA-mediated toxic effect of EF and
LF(12, 13) . Trypsin-activated (``nicked'')
PA, consisting of PA
complexed with the complementary
fragment, PA
, is capable of forming well-defined,
cation-selective channels when added to planar lipid bilayer
systems(14) . Also, cells preloaded with
Rb
release this isotope when exposed to
PA
or nicked PA followed by a low pH pulse(16) .
forms high
molecular weight, dodecyl sulfate-resistant oligomers, which appear
predominantly as heptameric rings by electron microscopy(18) .
Similar or identical oligomers are generated in a time-dependent manner
in cells incubated with PA, and oligomer formation in vivo appears to require exposure of PA
to acidic pH,
presumably in an acidic intracellular compartment. Thus, inhibitors of
internalization or endosome acidification block conversion of
cell-associated PA
to high molecular mass species, and
acidification of the medium induces oligomer formation either in the
presence or absence of these inhibitors. The conditions required for
PA
oligomerization therefore correlate with those required
for channel formation and translocation of EF and LF across cellular
membranes, suggesting that the oligomer may play a role in both
activities.
oligomer functions in
translocation of EF and LF, as hypothesized, its interaction with these
proteins might affect either the formation of PA
ion
channels or the conductance properties of the channels once formed.
Here we report that LF inhibits the PA
-induced
permeabilization of the plasma membranes of CHO-K1 cells under acidic
pH conditions and present data relevant to the mechanism of the
inhibition.
Toxin Purification
PA and LF were purified from
the Sterne strain of B. anthracis, according to the method of
Leppla (19) , with modifications, as described
previously(14) . The LF fragment, comprising the
amino-terminal 255 residues of LF, was cloned from the toxin plasmid
(pXO1) of the Sterne strain (20) of B. anthracis by polymerase chain reaction, with appropriate restriction sites
introduced by the 3` (NdeI) and 5` (BamHI) polymerase
chain reaction primers. The polymerase chain reaction product was then
cloned into an Escherichia coli expression vector, pET15b
(Novagen, Inc, Madison, WI), replacing the NdeI-BamHI
fragment. The recombinant protein was produced with an amino-terminal
hexa-histidine tag, allowing protein to be purified by
Ni
-chelate affinity chromatography. Briefly, cultures
were grown in Luria broth-ampicillin (100 µg/ml) to an OD
of 0.6-1.0, and protein expression was induced by
addition of isopropyl-1-thio-
-D-galactopyranoside (1
mM). Cell lysates were prepared by sonication, cleared by
centrifigation, and loaded onto a Ni
-charged
column(21) . The column was washed, and the protein was eluted
with imidazole according to the manufacturer's protocol. The
eluted protein was further purified by anion-exchange chromatography
(Mono-Q column on the fast proteim liquid chromatography system,
Pharmacia), yielding 1-2 mg of purified protein/liter of culture.
The purity of all proteins used in the experiments was greater than
95%, as judged by SDS-polyacrylamide gel electrophoresis and Coomassie
Blue staining. Protein concentrations were determined using the
Bradford reagent (Pierce). Nicked PA was produced by treatment of PA
(0.3 mg/ml = 3.6 mM) with trypsin (1 µg/ml) for 45
min at 37 °C, and the reaction was stopped by adding soybean
trypsin inhibitor to 10 µg/ml.
Cell Culture
The Chinese hamster ovary cell line
(CHO-K1), from the American Type Culture Collection, was grown in
Ham's F-12 medium supplemented with 10% calf serum, 500 units/ml
penicillin G, and 500 units/ml streptomycin sulfate (Life Technologies,
Inc.).
CHO-K1 cells were plated at a density of 10Rb
Efflux
Measurements
cells/well in 6-well culture plates or 2
10
cells/well in 24-well culture plates, depending on the
experiment. Approximately 10-12 h after plating, fresh medium
containing
Rb
(1 µCi/ml, NEN DuPont)
was added, and the cells were incubated for an additional 9-10 h.
PA binding was carried out by incubation of cells with fresh medium
containing 6 nM nicked PA for 2 h at 4 °C. The cells were
then washed two times with cold Dulbecco's phophate-buffered
saline (PBS) (Sigma) to remove unbound PA and treated with isotonic
buffers (20 mM MES-Tris, 145 mM NaCl, pH
4.0-7.0) to initiate the
Rb
release
assay. After 30 min at 4 °C, the medium was removed, and released
Rb
was measured using a
counter
(LKB-Wallac Clinigamma). Finally, SDS (1%) was added to the cells to
determine cell-retained
Rb
. For the
kinetic study of
Rb
release, the cells
were pulsed with pH 5.0 buffer, and an aliquot of medium was removed at
intervals for isotopic assay. All experiments were performed in
duplicate.
Rb
was calculated as follows: %
Rb
release = (released
Rb
)/(total
Rb
[released + retained])
100. Spontaneous
release was determined in the absence of PA and was subtracted from the
reported values. Spontaneous release of
Rb
was approximately 20% for cells treated with pH 5.0 buffer for 30
min.
Detection of PA
CHO-K1 cells were seeded at a density of 2
Oligomerization in Cells by
Immunoblotting
10
cells/well in 24-well culture plates. After 24 h
the plate was placed on ice, and binding of nicked PA (6 nM)
was carried out as described above at 4 °C for 2 h. Unbound PA was
removed by washing cells with cold PBS three times. The cells were
treated with isotonic buffers at different pH values (0.4 ml/well). For
analysis of the effect of LF on PA
oligomerization, LF
(0.1 µM) was included in these buffers. After 20 min the
buffer was removed, and stop solution (PBS plus 1% Triton X-100 and 1%
SDS) was added. The samples were then loaded on a 7.5%
SDS-polyacrylamide gel, and after electrophoresis the proteins were
transferred to a nitrocellulose membrane. The membrane was incubated
with rabbit anti-PA antibody in TBS (10 mM Tris-HCl, 150
mM NaCl, pH 7.5) containing 5% milk. After washing three time
with TBST (TBS plus 1% Triton X-100) a secondary antibody (horseradish
peroxidase-conjugated anti-rabbit IgG from Sigma) was added in TBS
containing 5% milk. PA was visualized using the DuPont Renaissance Kit
according to the manufacturer's protocol. The relative amounts of
PA
monomer and oligomer were calculated from the intensity
of protein bands after densitometric scanning (LKB Ultroscan).
CHO-K1 cells (2.5 Na
Influx
Measurements
10
cell/well)
were plated in 24-well culture plates 24 h prior to the start of an
experiment. Nicked PA (6 nM) was incubated with cells for 2 h
at 4 °C, and cells were washed with cold PBS. The cells were
treated with low pH (5.0) medium for 30 min at 4 °C to induce ion
channel formation, and LF (0.2 µM) was then added into the
low pH medium for an additional 30 min at 4 °C. An equal volume of
the same low pH medium containing 2 µCi/ml
Na
(NEN Dupont) was added at time 0, the
incubation was continued at 4 °C, and at intervals the medium was
removed from selected samples and the cells were washed four times with
cold PBS. The cell-associated radioactivity was determined after
dissolving the cells in 1% SDS. Spontaneous influx of
Na
determined in the absence of PA has
been subtracted from the values reported. In a parallel experiment
cell-associated protein was determined at intervals with Bradford
reagent. All experiments were performed in duplicate.
Rb
efflux.
Briefly, CHO-K1 cells preloaded with
Rb
were incubated with trypsin-activated PA [nicked PA
(PA
+ PA
); 6 nM] for 2 h
at 4 °C, unbound toxin was removed by washing, and medium buffered
at low pH (pH 5.0) was added. Release of
Rb
from the cells was then quantified at intervals by assaying
aliquots of the medium. Upon addition of pH 5.0 isotonic buffer,
Rb
efflux began immediately, continued
linearly for the first 10 min, and was complete by 15-20 min, at
which point 70-80% of total cell-associated
Rb
had been released (Fig. 1).
Inclusion of LF (0.1 µM) in the acidic pulse buffer caused
a rapid and strong inhibition (
5-fold) of the rate of
Rb
release (Fig. 1).
Rb
release continued at an approximately
constant rate over the 30-min period of the experiment in the presence
of LF. A more gradual decline in the rate of
Rb
release was seen when LF was added 1, 2, or 5 min after the low
pH buffer. LF
(residues 1-255), the PA-binding domain
of LF, had a similar inhibitory effect, whereas bovine serum albumin or
lysozyme caused no inhibition (Table 1). Tetrahexylammonium
bromide (HeNBr) also inhibited
Rb
release, consistent with earlier reports (14, 16) .
Rb
efflux from CHO-K1 cells by LF. CHO-K1
cells pre-loaded with
Rb
in 6-well
culture plates were incubated with nicked PA (6 nM) for 2 h at
4 °C. Unbound PA was removed by washing with cold PBS, and low pH
(5.0) isotonic buffer was added in the presence (
) or absence
(
) of LF (0.1 µM). Alternatively, LF was added after
exposure of cells to low pH medium for 1 (
), 2 (▪), or 5
(▴) min, as indicated by arrows, to a final
concentration of 0.1 µM. At the times indicated an aliquot
of medium was removed to determine released
Rb
.
Rb
release by LF or LF
was
dependent both on the concentration of the inhibitory proteins and on
the pH. Half-maximal inhibition was seen at
40 nM LF or
100 nM LF
when these proteins were included
in the pH 5.0 pulse medium (Fig. 2). The inhibition by LF was
maximal at or near this threshold pH and decreased as the pH was
lowered toward pH 4.0 (Fig. 3). A similar dependence on pH was
seen with LF
, but the reduction in inhibitory activity with
decreasing pH was less pronounced (not shown).
Rb
release by LF and LF
.
CHO-K1 cells pre-loaded with
Rb
in
24-well culture plates were incubated with nicked PA (6 nM) at
4 °C for 2 h. Unbound PA was removed by washing with cold PBS, and
low pH (5.0) buffer containing the indicated amount of LF (
) or
LF
(
) was added. After a 30-min exposure to the acidic
buffer at 4 °C, aliquots of medium were removed to measure released
Rb
.
Rb
release by LF. CHO-K1 cells pre-loaded
with
Rb
in 24-well culture plates were
incubated with nicked PA (6 nM) for 2 h at 4 °C. Unbound
PA was removed by washing with cold PBS, and pH (4.0-7.0) buffers
were added (
). After 30 min the medium was removed for
determination of released
Rb
. To
determine the effect of LF on
Rb
release,
LF (0.1 µM) (
) was included in the pH (4.0-5.5)
buffers.
Rb
-loaded CHO-K1 cells
to which PA had been bound were pulsed with pH 4.5 buffer for 1 min to
induce ion channel formation, and after removal of the buffer, the
cells were treated immediately with LF in buffers of various pH. The
inhibition of
Rb
release by LF was
maximal at pH 5.5 and declined as the pH was either raised or lowered
from this value (Table 2). Control experiments (not shown)
indicated that neutralization of the medium did not affect the kinetics
of
Rb
release after channel formation by
nicked PA at low pH.
Rb
release by LF and LF
in the experiments reported
above could be attributable to effects on channel formation, on ion
conductance by the channels, or on both. To distinguish among these
possibilities, we first examined the effects of LF on the formation of
high molecular mass, dodecyl sulfate-resistant oligomers of
PA
, which are believed to represent the ring-shaped
heptamers seen by electron microscopy and to be responsible for the
PA
channels in membranes(18) . Cells that had been
incubated with nicked PA were treated with buffers of various pH for 20
min and then disrupted with dodecyl sulfate. Analysis of the extracts
by SDS-polyacrylamide gel electorphoresis and Western blotting revealed
oligomer in samples treated at pH 5.25 or lower (Fig. 4), which
correlates with the pH dependence profile of channel formation.
Inclusion of LF in the low pH buffers caused no apparent change in
oligomer formation.
oligomerization as a function of pH. CHO-K1 cells in 6-well culture
plates were incubated in the presence of nicked PA (6 nM) at 4
°C for 2 h, and unbound PA was removed by washing with cold PBS.
The cells were treated with the pH (4.0-7.0) buffers indicated at
the top of the figure for 20 min (panel A, top).
Alternatively, LF (0.1 µM) was included in the pH buffers (panel A, bottom). At the end of incubation, the
buffer was removed, and the cells were dissolved in PBS containing 1%
SDS. The cell extract was electrophoresed on a polyacrylamide gel,
followed by Western blotting. The positions of the PA
monomer and oligomer are indicated. The plot in B shows
the percentage of oligomer formed in the presence (
) or absence
(
) of LF, as determined by densitometry of the blot in A.
oligomerization and the effect of LF on oligomerization. CHO-K1
cells in 6-well culture plates were incubated in the presence of nicked
PA (6 nM) for 2 h at 4 °C. After unbound PA was removed by
washing with PBS, the cells were exposed to a low pH buffer (pH 5.0, top panel, or pH 4.6, bottom panel) for the indicated
times. The reaction was stopped by removing the low pH buffer and
adding PBS containing 1% SDS. The cell extract was then processed for
Western blotting as described in Fig. 4. Percentage of oligomer
was calculated from densitometer scans of the blot (
). To
determine the effect of LF on PA
oligomerization, LF (0.1
µM) was added in the low pH buffer (
).
Alternatively, LF (0.1 µM) was included in the PA-binding
medium and was also present in the low pH buffer
(▪).
Rb
release
produced by LF, most of the inhibition appeared to be due to an effect
on ion conductance of the PA
channels. To support this
hypothesis we performed an experiment in which PA-bound cells were
first incubated with pH 5 medium for 30 min to allow channel formation
to proceed to completion. After an additional 30-min incubation in the
presence or absence of LF,
Na
was added,
and its influx was measured at intervals by assaying cell-associated
radioactivity. As shown in Fig. 6, a strong inhibition by LF was
seen, supporting the conclusion that binding of LF to preformed
PA
channels significantly affected their ion conductance.
Na
into CHO-K1 cells. CHO-K1 cells in
24-well culture plates were incubated with nicked PA (6 nM)
for 2 h at 4 °C. After unbound PA was removed by washing with cold
PBS, the cells were treated with low pH (5.0) buffer for 30 min, and
the incubation was continued in the presence (
) or absence
(
) of LF (0.2 µM) for an additional 30 min at 4
°C.
Na
was then added, and
cell-associated radioactivity was determined at intervals as described
under ``Experimental Procedures.'' Cellular protein,
determined with Bradford reagent in parallel sets of samples in the
absence of
Na
, showed no loss of cells
during the course of the experiment.
-mediated
Rb
release from CHO-K1 cells under acidic
pH conditions strongly suggests that LF interacts directly with
PA
channels in the plasma membrane. (EF would presumably
cause a similar inhibition, but we were unable to test it because of
difficulties in obtaining sufficient quantities of the protein.) The
interaction of LF with the PA
channels is consistent with
the known role of PA in membrane translocation of EF and LF and is
supported by earlier results showing binding of LF to PA
in vivo and in vitro. EF and LF have been found
to bind to cells only after PA has attached and been proteolytically
processed (by the endogenous protease, furin(10) ), and
sedimentation analysis and native polyacrylamide gel electrophoresis
have confirmed a direct interaction in vitro(4) .
showed similar inhibitory
effects on PA
ion channel activity ( Fig. 2and Table 1) indicates (i) that the PA-binding portion of LF is
largely responsible for the observed inhibition and (ii) that the
EF/LF-binding site on PA
is intact and exposed after
PA
inserts into the plasma membrane. While the
COOH-terminal portion of LF (residues 256-776) is apparently not
required for the inhibition of PA
ion channel activity,
its presence decreases the inhibition at lower pH values, perhaps due
to influences on conformation of the LF
moiety. Our data on
the inhibition of PA
channel activity by LF are consistent
with the report by Finkelstein (22) that addition of EF or LF
to PA
channels in planar bilayers at acidic pH causes a
reduction of conductance on a time scale measured in many seconds.
Recently Finkelstein and co-workers have found that the ion conductance
of PA
is attenuated by EF and LF at the single-channel
level, (
)which is consistent with our conclusions regarding
the inhibition by LF and LF
.
has been reported,
from results obtained with a soluble competitive binding assay
utilizing a monoclonal antibody against PA(4) . Competitive
displacement of radiolabeled LF from macrophage-associated PA at pH 7.5
occurred at a value of
2.4 nM(23) . These results
suggest that the affinity of LF for PA
is strong, in the
nanomolar to picomolar range, but further work will be required to
obtain precise values of the affinity constant under various
conditions.
oligomerization and ion channel formation in the plasma
membrane of CHO-K1 cells under acidic pH conditions(18) . The
conditions required for oligomerization and channel formation by
PA
correlate with those required for the translocation of
LF and EF across cellular membranes(18) , and we infer that the
PA
oligomer is likely to be the structural entity that
mediates both translocation and ion conductance. Although the presence
of LF did not prevent PA
oligomer formation in the plasma
membrane, it did slow the rate of oligomerization somewhat at pH 5.0 (Fig. 5). This could be attributed to LF-bound PA
undergoing self-association more slowly than free PA
monomers, or to PA
needing to dissociate from LF
prior to oligomerization. The fact that LF had little or no effect on
PA
oligomerization in the plasma membrane of CHO-K1 cells
at pH 4.6 and the finding that LF can efficiently inhibit PA
ion channel activity at this pH suggest that PA
can
undergo oligomerization in cells with or without LF bound. These
results also suggest that the LF-binding site on the PA
molecule is accessible before, during, or after its
oligomerization and insertion into the plasma membrane of CHO-K1 cells.
channel. The complex of PA
LF or
PA
EF may insert into the endosomal membrane, before
or after oligomerization, and the channel formed by PA
may
then mediate transfer of unfolded LF and EF to the cytosol, where they
would then refold into native form. In the second model, monomeric
PA
is assumed to mediate translocation. The acidic
environment of the endosome may trigger insertion of
PA
LF or PA
EF complexes into the
endosomal membrane, resulting in transfer of LF and EF to the cytosol
via the lipid phase per se or the PA
-lipid interface.
According to this model, oligomerization of PA
and channel
formation are secondary events in which, after the translocation,
lateral diffusion of the membrane-inserted PA
monomers
results in their oligomerzation and channel formation. The ability of
LF to inhibit channel activity under acidic pH conditions favors the
former model.
, which partially occludes the channel and inhibits ion
transport. The binding may be within the PA
channel (which
may correspond to the lumen of the heptameric PA
rings)
and thereby physically occlude the lumen, or it may be at peripheral
sites and attenuate conductance via conformational alterations induced
in PA
. No data are yet available from crystallography or
electron microscopy to localize LF or LF
bound to the
PA
oligomers. At pH 7.0 LF has only a slight inhibitory
effect on the activity of the PA
channel (Table 2),
suggesting that LF weakly interacts with PA
at neutral pH.
This finding may be relevant to the mechanism by which LF is released
from the channel and is able to enter the cytosol, which is near
neutral pH.
-dependent
Rb
release similar to that seen with LF.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.