From the Departments of ** Medicine, Pharmacology, and
§ Physiology and Biophysics, Case Western Reserve
University, Cleveland,Ohio 44106 and the ¶ Department of
Pathology, University of Michigan Medical School, Ann
Arbor, Michigan 48109
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ABSTRACT |
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Recent studies have demonstrated that the anti-apoptotic proteins, Bcl-2 and Bcl-xl, with the carboxyl-terminal hydrophobic domain removed, form cation-selective channels in the lipid bilayer reconstitution system. However, the regulatory properties of these channels are unknown. In this study, we investigated the ion-conducting properties of full-length Bcl-xl in the lipid bilayer reconstitution system. Our findings indicate that Bcl-xl forms a cation-selective channel that conducts sodium but not calcium and that Bcl-xl channel activity is reversibly inhibited by luminal calcium with a half-dissociation constant of ~60 µM. This calcium-dependent regulation of the Bcl-xl channel provides new insights into the roles of calcium and Bcl-2-related proteins in the programmed cell death pathway.
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INTRODUCTION |
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Bcl-2 and its related proteins are critical regulators of programmed cell death or apoptosis (1, 2). Composed of both anti- and pro-apoptotic members, this family of proteins functions in a programmed cell death pathway common to most multicellular organisms. It is generally believed that the ratio of death antagonists, such as Bcl-2 and Bcl-xl, and death agonists, such as Bax and Bcl-xs, plays a major role in the fate of the cell following an apoptotic stimulus (3). The anti-apoptotic proteins have also been suggested to function by interacting with caspases/CED-4 homologs (4, 5). Most of the Bcl-2-related proteins contain a hydrophobic carboxyl-terminal sequence that anchors the protein to membranes of organelles, including the endoplasmic reticulum (ER),1 mitochondria and nucleus, which play important roles in apoptosis (6-9). Despite their importance in regulating development and homeostasis in multicellular organisms, the exact physiological function(s) of these proteins remain elusive.
One of the current theories is that Bcl-2 family members regulate ion fluxes. This idea is supported by evidence that Bcl-2 overexpression prevents calcium redistribution from the ER to mitochondria following growth factor withdrawal (10) and inhibits apoptosis-associated calcium waves (11) and nuclear calcium uptake (12). Bcl-2 overexpression has also been shown to enhance the uptake of calcium by mitochondria (13) and preserve mitochondrial transmembrane potential (14). Our previous data have demonstrated that Bcl-2 overexpression is associated with a reduction in the transient elevation of cytosolic calcium induced by thapsigargin-mediated ER calcium ATPase inhibition (15). Furthermore, Bcl-2 overexpression maintains calcium homeostasis and calcium-dependent protein processing in the ER of thapsigargin-treated cells (16).
Recently, the x-ray and NMR structure of Bcl-xl was shown to resemble the physical structure of ion channel-forming bacterial toxins, such as diphtheria toxin and colicin Ia (17). Subsequently, Bcl-xl, Bcl-2 and Bax, have been shown to form ion channels using the lipid bilayer reconstitution system (18-21). However, these studies all utilized proteins from which the carboxyl-terminal hydrophobic domain had been deleted. Previous studies of the channel forming bacterial toxin, colicin Ia, showed that the channel formed by protein which lacks the hydrophobic transmembrane domain differs in conductance from the one formed by the intact colicin Ia (22). Thus, it is presently unclear whether the ion channel forming properties of the truncated Bcl-2 family members necessarily correspond to those of the intact proteins. Furthermore, it seems paradoxical that Bcl-2 and Bcl-xl, which function to inhibit apoptosis, should structurally and functionally resemble bacterial toxins whose main purpose is to form pores in cell membranes and ultimately destroy cells by disrupting ion homeostasis. This suggests that the ability of Bcl-2 and Bcl-xl to promote cell survival may not derive solely from their ability to form a channel. An explanation to this paradox may lie in understanding how Bcl-2 and Bcl-xl channel activity is regulated.
In light of our recent evidence that depletion of cellular calcium abrogates the anti-apoptotic effect of Bcl-2 (16), we set out to examine whether calcium is able to regulate the channel activity of anti-apoptotic Bcl-2 family members in a lipid bilayer reconstitution system. In the present report, we show that full-length Bcl-xl, produced in a bacterial expression system, forms a monovalent cation-selective channel that conducts sodium but not calcium. Significantly, we observed that Bcl-xl channel activity is inhibited by calcium, indicating for the first time that Bcl-xl forms a calcium-regulated cation channel.
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MATERIALS AND METHODS |
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Plasmid Preparation-- A full-length human bcl-xl cDNA (23) was polymerase chain reaction-amplified, using flanking primers (forward = 5'-CCCCCTTCTAGAATGTCTCAGAGCAACCGG-3' and reverse = 5'-GGGGCATGCCTCGAGTCATTTCCGACTGAAGAGTGAGCC-3', start/stop codons are underlined) to introduce a XbaI site in the 5' end and a XhoI site in the 3' end. The XbaI and XhoI-digested, gel-purified polymerase chain reaction product was subcloned into the pProex-1 expression vector (Life Technologies, Inc.) containing a 6-histidine (6×His) sequence, which allows affinity purification of expressed 6×His-Bcl-xl fusion protein. The entire bcl-xl cDNA sequence was identical to the published sequence with the exception of two conservative changes, A70G and V152A.
Protein Purification--
A single colony of Escherichia
coli, transformed with the full-length bcl-xl cDNA,
was cultured at 37 °C in LB medium with 100 µg/ml ampicillin.
Induction was carried out at an A600 of 0.7 with
1 mM isopropyl--D-thiogalactopyranoside at
37 °C for 5 h before harvesting cells by centrifugation. The
cells were pelleted and resuspended in 2-5 volumes of sonication
buffer (50 mM Na3PO4 (pH 8.0), 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride). Following sonication (1-min bursts/1-min cooling/200-300 watts), the cells were centrifuged at 20,000 × g for
1 h at 4 °C. The pellet was resuspended and solubilized as
described previously by Loo and Clarke (24) with minor modifications.
Briefly, the pellet was resuspended in 0.3 ml of buffer A (50 mM Na3PO4 (pH 8.0), 500 mM NaCl, 50 mM imidazole, and 20% (v/v)
glycerol). The proteins were then solubilized at 4 °C by adding 1 ml
of solubilization buffer (buffer A + 1% (w/v) CHAPS). Insoluble
material was removed by centrifuging at 16,000 × g for
15 min. The supernatant was applied to the nickel-nitrilotriacetic acid
column that was pre-equilibrated with 0.5 ml of nickel-agarose (Qiagen)
in buffer B (buffer A + 0.1% (w/v) CHAPS). The column was washed twice
with 0.6 ml of buffer B and with 0.6 ml of buffer C (10 mM
Tris-Cl (pH 7.0), 500 mM NaCl, 80 mM imidazole
(pH 7.0), 0.1% (w/v) CHAPS, and 20% (v/v) glycerol). The
6×His-tagged Bcl-xl proteins were eluted with 0.25 ml of buffer D (10 mM Tris-Cl (pH 7.0), 500 mM NaCl, 300 mM imidazole (pH 7.0), 0.1% (w/v) CHAPS, and 20% (v/v)
glycerol) in multiple fractions. Typically, cells from 100 ml of
culture yielded 0.5-3.0 µg of Bcl-xl protein. Purified proteins were
then characterized by SDS-PAGE (12.5% gels), followed by silver
staining (Bio-Rad) and Western blotting.
Western Blot Analysis--
Immunoblots were performed as
described previously (16). Briefly, full-length 6×His-tagged Bcl-xl
proteins were mixed with the sample buffer (200 mM Tris-Cl
(pH 6.7), 10% SDS, 5% -mercaptoethanol, 15% glycerol, 0.01%
bromphenol blue) and separated on a 12.5% linear SDS-PAGE. The
proteins were then transferred to a nitrocellulose membrane and blotted
with the Bcl-xl polyclonal antibody (Santa Cruz Biotechnology) and
horseradish peroxidase-linked secondary antibody using the ECL
detection system (Amersham Pharmacia Biotech).
Planar Lipid Bilayer Preparation and Single Channel Recordings-- Planar lipid bilayer membranes were formed across an aperture of 200 µm diameter with a mixture of phosphatidylethanolamine/phosphatidylserine/cholesterol in a ratio of 5:5:1 as described previously (25). Single channel currents were recorded with an Axopatch 200A patch clamp unit, and data analyses were performed with pClamp Software. The eluted fraction containing the purified Bcl-xl protein was either used directly or concentrated by Centriplus-30 (Amicon) and reconstituted in liposomes (phosphatidylethanolamine/phosphatidylserine/cholesterol in a ratio of 1:1:1) for the single channel measurements. The two preparations gave similar results. Purified Bcl-xl (60-100 ng) or Bcl-xl containing liposome was added to the cis-solution in the presence of asymmetric NaCl conditions at pH 7.4. The experiments were performed at room temperatures (23-25 °C). Single channel currents were recorded at 1 ms/point with the cut-off filter frequency set at 1 KHz.
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RESULTS |
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The full-length Bcl-xl protein was expressed in E. coli
following isopropyl--D-thiogalactopyranoside induction
and purified to homogeneity using nickel-nitrilotriacetic acid affinity
chromatography. The Western blot shown in Fig.
1A represents the elution
profile from the nickel column. The purified Bcl-xl has an apparent
molecular mass of ~31 kDa, which corresponds to the endogenous Bcl-xl
(data not shown). It has been shown previously that Bcl-xl runs
slightly higher than the predicted molecular mass of 26 kDa for the
full-length Bcl-xl protein (18). Silver staining of an eluted fraction
revealed a single band, indicating that the preparation was >95% pure
(Fig. 1B.)
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To characterize the single channel function of full-length Bcl-xl,
purified protein was reconstituted in the lipid bilayer, and the
selected current traces shown in Fig. 2
were recorded with 200 mM NaCl present in the
cis-solution (to which the Bcl-xl protein was added) and 50 mM NaCl present in the trans-solution. Bcl-xl
displays channel activity, producing a larger current at +40 mV than
that at 40 mV (Fig. 2A). In addition, the current-voltage relationship has a reversal potential of
30 mV (Fig. 2B),
which corresponds to a selectivity ratio of
PNa/PCl = 13.9. These
findings indicate the cation-selective nature of the Bcl-xl channel. In separate experiments with a recording solution consisting of 200 mM NaCl (cis)/200 mM KCl
(trans), the full-length Bcl-xl channel had a reversal
potential of ~
5 to 0 mV, which corresponds to a relative
selectivity ratio of
PNa/PK = ~1-1.2. This
value is similar to the truncated Bcl-xl channel reported by Minn
et al. (18). A characteristic feature of the Bcl-xl channel
is the appearance of multiple conductance states, as shown in Fig.
2C. Typically, six distinctive conductance levels could be
identified, with each conductance level of ~50 picosiemens. The
association of multiple conductance states with the full-length Bcl-xl
channel is similar to that of the Bcl-xl channel that had the
carboxyl-terminal hydrophobic domain deleted (18). This characteristic
may be because of the propensity of Bcl-xl proteins to interact with one another, as this prevents us from distinguishing the current measurements from either single channels or multiple channels. At the
present, we do not know the stoichiometry of the Bcl-xl channel,
i.e. the number of subunits that participates in the formation of the cation-selective channel.
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To confirm that the conductance shown in Fig. 2 was a property of the Bcl-xl protein, we measured its conductance in the presence of the Bcl-xl polyclonal antibody, which recognizes first 19 amino acids of the amino terminus. As shown in Fig. 3, addition of ~31 nM antibody to the cis-solution produced a significant reduction in Bcl-xl channel activity (n = >3), whereas addition of an equivalent amount of bovine serum albumin to the cis-solution was without effect (data not shown). Furthermore, when the antibody was added to the trans-solution, no change in channel activity was observed. These findings suggest that the Bcl-xl channel activity may be regulated by protein-protein interactions at the amino terminus and confirm that the channel activity observed in the planar lipid bilayer system was indeed produced by Bcl-xl rather than any minor contaminants in the protein preparation. In addition, these data confirm that the protein is oriented in the cis-cytoplasmic trans-luminal manner.
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To study whether the full-length Bcl-xl channel is regulated by
calcium, 100 mM CaCl2 was added to the
trans-solution. The current traces shown in Fig.
4 were obtained with a NaCl gradient of
200 (cis)/50 mM (trans). Following
the addition of 100 mM CaCl2 to the
trans-solution, the activities of the channel at both +60 mV
and 60 mV were reduced to essentially zero, indicating that calcium
inhibited the function of the Bcl-xl channel. Furthermore, because an
inward current was not observed at
60 mV, the Bcl-xl channel does not
appear to be permeable to calcium.
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The dose-response relationship between luminal calcium concentration and Bcl-xl channel activity was examined, to further understand the effect of luminal calcium. The results indicate that calcium attenuates both the current amplitude and opening probability of the Bcl-xl channel activity, with a Ki of 57.78 ± 26.14 µM (Fig. 5A). In addition, the inhibitory effect was because of the action of luminal calcium, as suggested by the following observations. First, adding calcium to the cis-solution had a lesser inhibitory effect on the Bcl-xl channel current amplitude and opening probability (Fig. 5A). Second, adding 10 mM MgCl2 to the trans-solution did not affect the channel activity (Fig. 5A). In addition, the inhibitory effect of the Bcl-xl channel activity by luminal calcium was readily and consistently reversed by adding EGTA to the trans-solution (n = 5), as illustrated in Fig. 5B.
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DISCUSSION |
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Our findings indicate that full-length Bcl-xl forms a monovalent cation selective channel, with conductance properties similar to those described previously using recombinant proteins from which the carboxyl-terminal hydrophobic domain had been deleted (18-21). Despite the evidence suggesting that Bcl-2-related proteins conduct ions, the relationship of this property to apoptosis induction or repression is presently unknown. In the present study, however, we show that Bcl-xl channel activity can be regulated by calcium, a finding that may provide new insights into the function of the Bcl-2 family in the apoptotic pathway.
Regulation of ion channels by calcium is a well documented phenomenon. For example, intracellular calcium regulates the rate of calcium-dependent inactivation of the voltage-gated L-type calcium channel as well as the open-closed transitions of a wide variety of potassium channels, such as Maxi-K(Ca) (26, 27). Also, changes in luminal calcium concentration are known to regulate the rate of calcium release from the sarcoplasmic reticulum vesicles of muscle cells, as well as the single channel opening probability of the ryanodine receptor/calcium release channels (28).
Thus, our observation that an anti-apoptotic protein such as Bcl-xl is regulated by luminal calcium is not unusual, yet novel as it may have a significant impact on how we view the function of these proteins in the apoptotic pathway. Although calcium has been implicated as a mediator of apoptosis, its exact role is enigmatic. However, the maintenance of a high level of calcium within the ER lumen is known to be essential for cellular functions, including translation (29), protein processing (30), cell division (31), and cell survival. Recently, Tsien and colleagues (32) demonstrated, with their "cameleons" fluorescent indicators, that the calcium concentration in the ER of individual cells is as high as 400 µM under physiological conditions. In addition, using ER-targeted, low calcium affinity aequorin reconstituted with coelenterazine n, the physiological concentration of calcium in the ER lumen is estimated to be between 500 and 600 µM in intact cells (33). Our findings indicate that luminal calcium in the high µM range inhibits Bcl-xl channel activity. This suggests that when the calcium concentration in the ER lumen is maintained at this level, the Bcl-xl channel remains in a closed state, supporting ion homeostasis.
Unlike the ER lumen, the free calcium concentration is relatively low
in mitochondria. For example, mitochondrial free calcium in a single
living cardiac myocyte is estimated to be in the high nanomolar range
(34). Our data suggest that calcium at this concentration would keep
the Bcl-xl channel open. However, what do Bcl-xl and its channel
activity in the mitochondria have to do with promoting cell survival?
Interestingly, a recent study by Shimizu and colleagues demonstrated
that Bcl-2 prevents loss of mitochondrial membrane potential ()
induced by 20 µM calcium in isolated mitochondria (35).
Their findings suggested that Bcl-2 maintained this
by mediating
proton efflux. However, at a higher calcium concentration (100-200
µM), Bcl-2 failed to mediate proton efflux, leading to
the collapse of the
. This is consistent with our findings
regarding the dose-response relationship between luminal calcium
concentration and Bcl-xl channel activity. Hence, it is likely that
Bcl-xl channel activity may be regulated by the luminal calcium
concentration of the organelle with which it is associated, thereby
contributing to its anti-apoptotic function. Based on the present
findings, whether Bax and other pro-apoptotic proteins can be regulated
by calcium merits immediate investigation.
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ACKNOWLEDGEMENTS |
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We thank George Dubyak, Anna-Liisa Nieminen, and Edmunds Z. Reineks for their critical review of this manuscript; David D. Friel, Keunmyoung Lee, Alvin Changco, Brian M. Davis, Huiling He, Karen S. McColl, George N. Partal, XiaoMei Qi, Nancy S. Wang, Mikko T. Unkila, and Bryan D. Zerhusen for helpful discussions; and Diane Baus for her help in silver staining.
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FOOTNOTES |
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* This work was supported by Training and Metabolism Grant 5T32DK07319, National Institutes of Health Grants RO1 CA42755 (to C. W. D.) and AG-15556 (to J. M.), and by an Established Investigatorship from the American Heart Association (to J. M.).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 (to J. M. or
C. W. D.): Dept. of Physiology and Biophysics, Dept. of
Medicine, Case Western Reserve University, 10900 Euclid Ave.,
Cleveland, OH 44106. For J. M.: Tel.: 216-368-6718; Fax:
216-368-1693; E-mail: jxm63{at}po.cwru.edu; for C. W. D.: Tel.:
216-368-1176; Fax: 216-368-1166; E-mail: cwd{at}po.cwru.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; 6×His, 6 × histidine; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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