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J Biol Chem, Vol. 275, Issue 16, 12321-12325, April 21, 2000
,
From the Department of Medical Genetics, Biomedical
Research Center, CREST of Japan Science and Technology Corp., the
¶ Department of Physiology, Osaka University Graduate School of
Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan, and the
§ Single Molecule Processes Project, International
Cooperative Research Project, Japan Science and Technology Corp.,
2-4-14 Senba-Higashi, Mino, Osaka 562-0035, Japan
 |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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The Bcl-2 family of proteins, consisting of
anti-apoptotic and pro-apoptotic members, regulates cell death by
controlling mitochondrial membrane permeability that is crucial for
apoptotic signal transduction. We have recently shown that some of
these proteins, such as Bcl-xL, Bax, and Bak,
directly modulate the mitochondrial voltage-dependent anion
channel (VDAC) and thus regulate apoptogenic cytochrome c
release and potential loss. To elucidate the molecular mechanisms of
VDAC regulation by Bcl-2 family proteins, an electrophysiological study
was carried out. It was found that VDAC and pro-apoptotic Bax created a
large pore, with conductance levels 4- and 10-fold greater than those
of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent
modulation of their activity, the VDAC-Bax channel had neither of their
properties. Anti-apoptotic Bcl-xL and its BH4 oligopeptide
completely closed the VDAC, in contrast to the Bax. Cytochrome
c passed through a single VDAC-Bax channel but not through
the VDAC or Bax channel in a planar lipid bilayer. These data provide
direct evidence that VDAC forms a novel large pore together with Bax.
Apoptosis is a tightly regulated cell death mechanism that is
required for selective elimination of cells. Various apoptotic signals
eventually converge to activate a family of cysteine proteases called
caspases, which then cleave a critical set of cellular proteins to
initiate apoptotic cell death. The Bcl-2 family of proteins is a well
characterized regulator of apoptosis that acts upstream of caspase
activation (1, 2). It consists of the following three distinct
subfamilies: 1) anti-apoptotic members, such as Bcl-2 and
Bcl-xL, with sequence homology at Bcl-2 homology 1 (BH1),1 BH2, and BH3 domains,
and in most cases BH4 domain as well; 2) pro-apoptotic members, such as
Bax and Bak, with sequence homology at BH1, BH2, and BH3; and 3)
pro-apoptotic proteins that only share homology at the BH3 domain
(BH3-only proteins), such as Bid, Bik, and Bim (1, 2). It has been
shown that in addition to BH1 and BH2, the BH4 domain is required for
anti-apoptotic activity of Bcl-2 and Bcl-xL and that the
BH3 domain of the pro-apoptotic members is essential and, itself,
sufficient for pro-apoptotic activity (1-3).
Recent evidence has shown that the mitochondria play a crucial role in
apoptosis by releasing the apoptogenic cytochrome c from the
inter-membrane space into the cytoplasm (1-3). Once in the cytoplasm,
cytochrome c activates a major apical caspase, caspase-9, in
concert with Apaf-1 and dATP (or ATP), and the activated caspase-9
subsequently activates an effector caspase, caspase-3 (4, 5). It has
been shown that Bcl-2 family proteins regulate cytochrome c
release in isolated mitochondria: pro-apoptotic Bax and Bak induce
cytochrome c release, whereas anti-apoptotic Bcl-2 and
Bcl-xL prevent the change (6-11). Recently, we have shown that some of the Bcl-2 family of proteins can bind directly to the VDAC
and modulate its activity (12). VDAC is a mitochondrial outer membrane
channel that usually functions as the pathway for the movement of
various substances to and from the mitochondria (13), and it is
considered a component of permeability transition pore complex that
plays a role in permeability transition (14, 15). Bax and Bak enhance
VDAC activity, whereas Bcl-xL inhibits it (12), although
the regulatory mechanisms still remain to be elucidated. We have also
shown that Bax and Bak induce the translocation of cytochrome
c through the VDAC in liposomes (12). To investigate the
detailed mechanism of VDAC regulation by the Bcl-2 family of proteins,
we employed electrophysiological techniques in the present study.
Chemicals--
Anti-human Bax (N20) polyclonal antibodies and
anti-human VDAC (31HL) monoclonal antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA) and Calbiochem (La Jolla, CA), respectively. Anti-pigeon cytochrome c monoclonal antibodies
(65971A), which cross-react with horse cytochrome c, were
from PharMingen (Tokyo, Japan). Cy3-labeling kit was from Amersham
Pharmacia Biotech. Other chemicals were obtained from Wako Biochemicals
(Osaka, Japan).
Protein Purification--
Human Bax and its mutants were
expressed as a His-tagged protein in Escherichia coli strain
XL1-blue using the Xpress System (Invitrogen), as described elsewhere
(7). Human Bcl-xL protein was expressed as glutathione
S-transferase fusion proteins in E. coli strain
DH5 The Electrophysiological Analysis--
The electrophysiological
study was performed as described previously (16). Briefly, a planar
lipid bilayer was formed across a 300-µm diameter aperture in a
Teflon wall. The bilayer-forming solution contained asolectin (20 mg/ml
n-decane). The test protein (1 µg/ml) was added to one
side (cis) of the bilayer, and the other side of the bilayer
(trans) was grounded. The symmetrical solution contained 100 mM NaCl, 20 mM Hepes-NaOH (pH 7.3), and 0.1 mM CaCl2, except in the Bcl-xL
experiment, which was carried out at pH 5.5 to facilitate incorporation
of Bcl-xL by the lipid membranes (12). The asymmetrical
solution was made by changing the salt concentration to 500:100
mM NaCl (cis:trans). Both the cis and
trans compartments were connected by a KCl agar bridge to
separate chambers with an AgCl2 electrodes. Data were
acquired and analyzed using pClamp software (Axon Instruments).
Immunoprecipitation and Western Blot Analysis--
Purified VDAC
(20 µg/ml) was incubated with rBax Cytochrome c Translocation Assay--
Horse cytochrome
c (1 mM) was labeled with Cy3 dye according to
the manufacture's protocol (Amersham Pharmacia Biotech).
Anti-cytochrome c antibody (0.4 µg) was applied to a glass
dish with a diameter of 5 mm for 1 h. After drying, 3% BSA (3 µl) was added. After 3 h, the indicated amount of Cy3-cytochrome
c or trans side solution was added for 1 h.
After drying and washing with phosphate-buffered saline, samples were
observed under a confocal fluorescence microscope (Zeiss, LSM410), and
the fluorescence intensity was measured.
Recently, we have shown that Bax and Bak enhance VDAC activity
through direct binding with VDAC (12). Enhancement of VDAC activity by
Bax/Bak can be explained in the following three ways: 1) Bax/Bak
enhances VDAC activity, 2) VDAC enhances Bax/Bak activity, or 3)
Bax/Bak and VDAC produce a novel channel. As we previously showed that
a VDAC inhibitor, polyanion, blocks Bax-mediated enhancement of VDAC
activity (12), the second possibility is rather unlikely. To obtain
some insights into a channel formed by VDAC and Bax/Bak, we performed
an electrophysiological study by measuring channel currents in a planar
lipid bilayer system with symmetrical and asymmetrical salt solutions.
Bax
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
and purified on a glutathione-Sepharose column and was released
from glutathione S-transferase by cleavage with thrombin
(12). All purified proteins were finally suspended in buffer composed
of 20 mM Hepes-K+ (pH 7.4) and 1 mM
dithiothreitol. Mock control proteins were prepared using glutathione
S-transferase- and His-tagged proteins from empty vectors.
Rat liver mitochondrial VDAC was purified as described previously
(12).
C and rBaxBH3C, and the
mixtures were immunoprecipitated with anti-VDAC antibody (Calbiochem;
31HL) and with normal mouse IgG, as described previously (12).
Co-immunoprecipitation of Bax was detected by Western blotting using
anti-Bax antibody (Santa Cruz; N20).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
C, lacking the C-terminal 20 amino acids, has been shown to form
an ion channel with weak cation selectivity and an elementary channel
conductance (conductance is expressed by S = A (current)/V
(voltage)) of 5.6 pS with long bursts of 26 and 250 pS in a planar
lipid bilayer at neutral pH (17). Similar results were also observed by
us (data not shown), although weak anion selectivity of the BaxC
channel has been reported elsewhere (18). We also found that highly
purified full-length Bax (Fig.
1A) also formed an ion
channel, which had its main elementary channel conductance below 20 pS
with a long burst of 210 ± 20 pS (n = 5) in 0.1 M NaCl at pH 7.2 (Fig. 1B). The VDAC formed an
ion channel with a single channel conductance of 600 ± 25 pS
(n = 6) in 0.1 M NaCl (Fig. 1, C
and D), which was consistent with the previous observations
of VDAC having a conductance of 4.5 nS in 1M KCl and 0.65 nS in 150 mM KCl (13, 20-22). When Bax was added to the
VDAC-incorporating planar lipid bilayer, a novel channel with a larger
conductance was formed (Fig. 1E), and it was never observed
in the absence of Bax. The conductance values of the single large
channel formed with VDAC and Bax were calculated to be 2.3 ± 0.1 nS (n = 8) in 0.1 M NaCl (Fig. 1,
E and I), about 4- and 10-fold larger than those
of the VDAC and the Bax channel, respectively. Interestingly, this
channel was almost continuously in an open state below 50 mV (open
duration, 44 h, and closed duration, 1.3 min, as determined from
the data of 7 independent similar experiments) (Fig. 1, E
and F). Because a channel with a similar conductance was
repeatedly generated only in the presence of both Bax and VDAC and
because the channel conductance occasionally showed discrete changes,
representing the transition from two VDAC-Bax channels (O1 + O1) to one channel (O1) and then back to two
channels (O1 + O1) (Fig. 1F), this
2.3 nS channel was not a nonspecific hole but a specific channel formed by the combination of Bax and VDAC. In some cases, we also observed a
channel with another conductance of 1.8 ± 0.1 nS
(n = 3) in the presence of Bax and VDAC (Fig. 1,
G and H (O2)). Unlike the 2.3 nS
channel, this channel with 1.8 nS conductance showed frequent current
fluctuations (Fig. 1G). The duration of the 1.8 nS channel (O2) observed was only 1.4 h, whereas the durations of
the 2.3 nS single (O1) and double (O1 + O1) channels were 42.3 and 1.7 h, respectively. The
nature of the 1.8 nS channel is still unknown, but it may represent
another VDAC-Bax channel or sublevels of the VDAC-Bax channel. Because
this 1.8 nS channel was not generated alone but only when the 2.3 nS
channel was formed, the 4.1 nS channel (O1 + O2) may represent a different conductance state of the
VDAC-Bax channel. Both the 2.3 and 1.8 nS channels were also observed
when BaxC was added to VDAC and VDAC was added to the Bax channel
(data not shown). Note that in the presence of VDAC, no formation of
Bax channel was observed (discussed below).
View larger version (24K):
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Fig. 1.
Bax and VDAC create a large
nonselective channel. A, analytical gel of purified
recombinant His-tagged Bax (left) and purified rat liver
VDAC (right) used in this study. Proteins (5 µg each) were
resolved on a 15% SDS-polyacrylamide gel electrophoresis gel and
stained with Coomassie Brilliant Blue. The molecular masses of the
marker proteins (lane 1) are indicated to the
left of the gel. B and C, recordings
of single channels induced by Bax or VDAC in planar lipid bilayers.
rBax (B) or VDAC (C) was added to the
cis side of a planar lipid bilayer in the presence of a 100 mM NaCl symmetrical solution (pH 7.4). Single channel
currents were measured at the indicated voltage. The closed state (zero
current) and the open state are marked by C and
O, respectively. The histogram in B shows two
main conductance levels of the Bax channel at -75 mV. D,
current-voltage plots of the VDAC. Data were obtained in the presence
of a 500:100 mM NaCl (cis:trans) asymmetrical
solution (pH 7.4). E-G, formation of a large channel in
planar lipid bilayers by a combination of Bax and VDAC. Bax was added
to the cis side of a VDAC-incorporating lipid bilayer in the
presence of 100 mM NaCl symmetrical solution (pH 7.4), and
the current was continuously monitored at the indicated voltage. The
zero current level (closed channel) and the open state are indicated by
C and O, respectively. The recording in
F shows that one of the two large VDAC-Bax channels
(O1 represents each of the channels) was transiently
closed (arrows). In G, two channels with
different conductances formed in the presence of Bax and VDAC are shown
(O1 and O2). H,
histogram showing two main conductance levels at 2.3 nS
(O1) and 1.8 nS (O2), which was
generated from the recordings presented in G. I,
current-voltage plots of Bax-VDACs with different conductances
(O1 and O2), demonstrating no ion
selectivity. The data were obtained in the presence of a 500:100
mM NaCl (cis:trans) asymmetrical solution (pH
7.4).
The VDAC-Bax channel showed significantly different properties from the VDAC and the Bax channel, as follows. As reported previously (13, 20-22), VDAC activity was modulated by the membrane potential, with the channel almost continuously open below 30 mV, whereas it showed frequent opening and closing above 30 mV (Fig. 1C). Bax channel activity was also reported to be enhanced when a negative potential was applied to the same cis side as the protein (17). In contrast, VDAC-Bax channel activity was never affected by the membrane potential under the conditions studied (Fig. 1F). To further characterize the VDAC-Bax channel, ion selectivity was assessed using asymmetrical NaCl solutions. The VDAC showed weak anion selectivity (Fig. 1D) and the Bax channel showed cation selectivity (data not shown), consistent with previous reports (13, 17, 20-22), whereas the VDAC-Bax channel with 2.3 nS conductance and the 1.8 nS channel both showed no ion selectivity (Fig. 1I). Furthermore, a VDAC inhibitor, DIDS (23, 24), did not influence the VDAC-Bax channel (data not shown), indicating that this channel had lost sensitivity to a VDAC inhibitor. These data indicated that the VDAC-Bax channel was substantially different from the Bax channel or VDAC and suggested that VDAC and Bax co-operated to form a functionally novel large pore.
The BH3 region of Bax has been shown to be essential for the activity
of pro-apoptotic Bcl-2 family members, including Bax and Bak (1-3).
Consistently, we previously showed that the BH3 region is essential for
Bak-induced apoptotic mitochondrial changes (7), as well as for
Bax-induced enhancement of VDAC activity (12). As shown in Fig.
2A, rBaxBH3C, which
lacked both the BH3 region (amino acids 55-74) and the C-terminal 20 amino acids of Bax, bound to VDAC to the same extent as did BaxC but
did not enhance VDAC activity in liposomes (12). These results
suggested that Bax can bind to VDAC via another site besides BH3 and
that VDAC is regulated via the BH3 region. To extend our studies, we also analyzed the channel properties of BaxBH3C and the
combination of BaxBH3C and VDAC in planar lipid bilayers. As
shown in Fig. 2B, BaxBH3C formed an ion channel with a
primary conductance of 220 ± 10 pS (n = 3) in 0.1 M NaCl, which was similar to that of the Bax channel (Fig.
1B). Like the Bax and BaxC channels, the BaxBH3C
channel showed weak cation selectivity and enhanced activity with a
negative potential applied to the same cis side as the
protein (data not shown). The only difference observed between the Bax
and BaxBH3C channels was in their opening probability: the Bax
and BaxC channels showed fast opening and closing (Fig. 1B), whereas the BaxBH3C channel tended to be
continuously open below 50 mV (Fig. 2B), being similar to
the VDAC-Bax channel. Thus, the BH3 domain seems to play an important
role in gating. The fact that BaxBH3C, like Bax, could form an
ion channel but could not effectively induce apoptosis might suggest
that the pro-apoptotic activity of Bax is not dependent solely on its
channel-forming ability. When BaxBH3C was added to a
VDAC-incorporating planar lipid bilayer, a larger conductance channel,
such as the 2.3 nS VDAC-Bax channel, was not formed (n = 5) (Fig. 2C), consistent with the observation that
BaxBH3C did not enhance VDAC activity in liposomes (12). The
cooperativity of Bax mutants with VDAC parallels their pro-apoptotic
activity, suggesting that the ability to form the VDAC-Bax channel
underlies the pro-apoptotic ability of Bax.
|
We also examined the effect of Bcl-xL on the VDAC in planar
lipid bilayers. As previously shown (12), VDAC activity is inhibited by
addition of rBcl-xL. As shown in Fig.
3A, addition of
rBcl-xL to the VDAC reduced the channel current to nearly
zero in a planar lipid bilayer. Although it has been shown that
Bcl-xL itself forms a channel (25),
Bcl-xL-specific channel was not observed. The lack of an
observation of formation of Bcl-xL (Fig. 3A) or
Bax channels (Fig. 1E) in the presence of VDAC was probably
due to higher affinity of Bcl-xL and Bax to VDAC than lipid
membranes.
|
Surprisingly, the BH4 oligopeptide (corresponding to amino acids 4-23 of Bcl-xL), when added at a concentration slightly higher than that of rBcl-xL, almost completely inhibited VDAC activity in a planar lipid bilayer (Fig. 3B), consistent with our previous observations with VDAC liposomes (26). This activity was not observed with BH4 mutant oligopeptides or with an oligopeptide from the corresponding region of Bak (data not shown). These results indicated that Bcl-xL completely closed the VDAC and suggested that the BH4 domain, which has been shown to be essential for the anti-apoptotic activity of Bcl-2 and Bcl-xL (27-31), has an intrinsic ability to cause VDAC inhibition.
Because we recently found that addition of Bax to VDAC-liposomes allows
cytochrome c to pass through the liposome membranes (12), we
examined whether cytochrome c could pass through a single
Bax-VDAC in a planar lipid bilayer. To measure the movement of
cytochrome c through the Bax-VDAC, Cy3-labeled cytochrome
c was produced and was detected by binding to
anti-cytochrome c antibody-coated dishes followed by
spectrophotometric detection. As shown in Fig.
4A, the amount of
Cy3-cytochrome c could be quantitatively measured from
105-1015 molecules. Cy3 itself was not
detected even at 1020 molecules (data not shown). When
Cy3-cytochrome c was added to the cis side of a
VDAC-Bax-incorporating planar lipid bilayer at 30 mV, a significant
increase of Cy3-cytochrome c was detected in solution on the
trans side in a time-dependent manner (Fig. 4B). Translocation of cytochrome c was roughly
calculated to occur at 10 molecules/s/channel. During the assay, the
VDAC-Bax channel was continuously open, and channel conductance
remained unchanged (Fig. 4C), excluding the possibility of
physical rupture of the lipid bilayer in these experiments. In
contrast, neither VDAC nor the Bax channel allowed Cy3-cytochrome
c translocation across a planar lipid bilayer (Fig.
4B). These results indicated that cytochrome c
could pass through a single VDAC-Bax channel.
|
Here, we have shown that the combination of Bax and VDAC forms a large nonselective channel. This channel was substantially different from VDAC and the Bax channel in that it did not show voltage-dependent modulation of activity and ion selectivity, so the VDAC-Bax channel seems to possess a novel function. The molecular structure of the VDAC-Bax channel is still unknown, but it might be a composite channel. We have previously shown using a liposome system that a large channel through which cytochrome c passes is formed only in the presence of both VDAC and Bax, even when the proteins are initially incorporated into the lipid membranes (12), excluding the possibility that VDAC merely facilitates incorporation of Bax into a planar lipid bilayer to form a large Bax channel.
The VDAC was reported to be 3 nm in diameter (19), which is similar to
the size of cytochrome c. Because the pore size of the
VDAC-Bax channel was 4-fold greater than that of the VDAC according to
our electrophysiological data, the VDAC-Bax channel should be large
enough for cytochrome c to pass through. Indeed, we were
able to detect cytochrome c translocation through this channel. In contrast, neither VDAC nor the Bax channel allowed Cy3-cytochrome c translocation across a planar bilayer.
There was no significant change of current through the channel even when cytochrome c passed through it, probably due to both
the large pore size and the low rate of cytochrome c
translocation. To completely understand the regulation of VDAC by the
Bcl-2 family of proteins, elucidation of the molecular structure of the
VDAC-Bax and VDAC-Bcl-xL channels is essential, but this
awaits the three-dimensional structural analysis.
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FOOTNOTES |
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* This study was supported in part by a grant for Scientific Research on Priority Areas, by a grant for Center of Excellence Research, and by a grant for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.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:
81-6-6879-3363; Fax: 81-6-6879-3369; E-mail:
tsujimot@gene.med.osaka-u.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: BH, Bcl-2 homology; VDAC, voltage-dependent anion channel; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; pS, picosiemens.
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RESULTS AND DISCUSSION
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