Address correspondence and reprint requests to Subhendu Ghosh, Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India. E-mail: firstname.lastname@example.org
Apoptosis is a controlled form of cell death that participates in development, elimination of damaged cells and maintenance of cell homeostasis. Also, it plays a role in neurodegenerative disorders like Alzheimer's disease. Recently, mitochondria have emerged as being pivotal in controlling apoptosis. They house a number of apoptogenic molecules, such as cytochrome c, which are released into the cytoplasm at the onset of apoptosis. When rat brain mitochondrial voltage-dependent anion channel (VDAC), an outer mitochondrial membrane protein, interacts with Bcl-2 family proteins Bax and tBid, its pore size increases, leading to the release of cytochrome c and other apoptogenic molecules into the cytosol and causing cell death. Regulation of this tBid- and Bax-induced increase in pore size of VDAC is a significant step to control cell death induced by cytochrome c. In this work, we have shown, through bilayer electrophysiological experiments, that the increase in VDAC conductance as a result of its interaction with Bax and tBid is reduced because of the action of cyclic AMP-dependent protein kinase A (PKA) in the presence of ATP. This indicates that the increase in the pore size of VDAC after its interaction with Bax and tBid is controlled via phosphorylation of this channel by PKA. This, we believe, could be a mechanism of controlling cytochrome c-mediated cell death in living cells.
direct inhibitor of apoptosis protein binding protein of low pI
second mitochondria-derived activator of caspases
protein kinase A
voltage-dependent anion channel
Apoptosis is known to take place in cells through two major pathways: the death receptor and the mitochondrial pathways (Budihardjo et al. 1999). In the latter, an increase in mitochondrial membrane permeability is a general observation. The release of pro-apoptotic substances, such as the apoptosis-inducing factor (Susin et al. 1999) endonuclease G (Li et al. 2001), Smac/DIABLO (Du et al. 2000; Verhagen et al. 2000) and cytochrome c (Adams and Cory 2001), is responsible for the mitochondria-mediated cell death. Of these, the cytochrome c-mediated pathway is significant in various physiological and pathological processes (Jiang and Wang 2004). However, less is known about its regulation. It has been reported that cytochrome c release plays a major role in neurodegenerative disorders, and regulation of its release has implications for the therapy of neurodegenerative diseases (Clayton et al. 2005).
Voltage-dependent anion channel (VDAC), an abundant protein in the outer mitochondrial membrane, plays a significant role in cytochrome c-mediated cell death (Shimizu et al. 1999, 2000; Banerjee and Ghosh 2004a). VDAC forms a large voltage-gated pore (2.5–3 nm) and acts as the pathway for the movement of substances in and out of the mitochondria by passive diffusion (De Pinto et al. 1985). Cytochrome c is released into cytosol through VDAC in the presence of the Bcl-2 family proteins Bax and tBid during apoptosis as a result of an increase in the pore size of VDAC. Release of cytochrome c from mitochondria inactivates the electron transport chain and causes cell death (Krippner et al. 1996). Also, release of cytochrome c is known to trigger caspase activation through Apaf1 (Li et al. 1997), which finally leads to cell death. It is still not clear how the normal physiological system makes sure that the leakage of cytochrome c into the cytosol does not occur. How do living cells resist the aforesaid process? When do the cells fail to offer this resistance and succumb to death? Regulation of VDAC might play an important role in this.
Various modes of regulation of VDAC have been reported to date. One of them suggests that, following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure (van der Heiden et al. 2001). Recently, we have shown that plasminogen protein interacts with VDAC and leads to closure of the channel, which might stop efficient exchange of metabolites across the mitochondrial outer membrane causing cell death (Banerjee and Ghosh 2004b). Similarly, glutamate (Gincel and Shoshan-Barmatz 2004) and NADH (Zizi et al. 1994) are also known to interact with VDAC and facilitate channel closure.
Among the many potential molecular mechanisms for regulating the activity of ion channels, protein phosphorylation plays a particularly important role (Catterall 1988; Levitan 1988, 1994). This is not surprising, given the central role of protein phosphorylation in a wide variety of cellular, metabolic and signaling processes (Cohen 1988, 1992; Hemmings et al. 1991). Regulation by phosphorylation is not restricted to one or another class of ion channels; many, and perhaps all, ion channels are subject to modulation by phosphorylation. Similarly, a number of different protein kinase signaling pathways can participate in the regulation of ion channel properties, and it is not unusual to find that a particular channel is modulated by several different protein kinases, each influencing channel activity in a unique way. We have demonstrated previously that VDAC can be phosphorylated in vitro by the catalytic subunit of protein kinase A (PKA) (Bera et al. 1995) and this reduces the channel current as demonstrated by bilayer electrophysiological data (Bera and Ghosh 2001). Here we have explored the role of phosphorylation of VDAC by cAMP-dependent PKA in the Bax- and tBid-induced cell death.
Materials and methods
Purification of voltage-dependent anion channel
VDAC was purified from rat brain mitochondria using the method of De Pinto et al. (1987). To check the purity of the VDAC, sodium dodecyl sulphate–polyacrylamide gel electrophoresis on a 12.5% polyacrylamide gel was performed. Recombinant full-length monomeric Bax and tBid were obtained from Professor Jean-Claude Martinou, Department de Biologie Cellulaire Sciences, Ernest-Ansermet, Geneva, Switzerland, as a gift.
Reconstitution of voltage-dependent anion channel in planar lipid bilayers and electrophysiological recording
VDAC was reconstituted into the planar lipid bilayers according to the method of Roos et al. (1982). Briefly, the apparatus consisted of a polystyrene cuvette (Warner Instrument Corp., Hamden, CT, USA) with a thin wall separating two aqueous compartments containing 500 mm KCl, 5 mm MgCl2 and 10 mm HEPES (pH 7.4). The polystyrene divider had a circular aperture with a diameter of 150 µm. Aqueous compartments were connected to an integrating patch amplifier (Axopatch 200A; Axon Instruments Molecular Devices Co., Sunnyvale, CA, USA) through a matched pair of Ag/AgCl electrodes. The cis chamber was connected to the head stage (CV-201) of the amplifier and the trans chamber was held at virtual ground. A 6 : 1 solution of DPhPC : cholesterol (obtained from Avanti Polar Lipids, Birmingham, AL, USA) dissolved in n-decane (obtained from Sigma Chemical Co., St Louis, MO, USA) was painted over the aperture to form the membrane. Reconstitution of VDAC in black lipid membrane (BLM) was initiated by adding 5 ng/mL protein (dissolved in 1% Triton X-100, 10 mm HEPES, pH 7.4) to the cis chamber. Axopatch 200A was connected to an IBM computer through an interface Digidata 1322A (Axon Instruments). After getting the single-channel recording of VDAC, the channel current was recorded using the acquisition software Clampex (pClamp 9.0, Axon Instruments). Single-channel recording of VDAC was performed in a symmetric bath solution. The solution in the cis chamber was then perfused out and fresh solution was added to avoid any further insertion of VDAC molecule.
Phosphorylation of voltage-dependent anion channel
VDAC was phosphorylated as described previously (Bera and Ghosh 2001). After single-channel recording of VDAC was performed, the following additions were made to the cis chamber: (i) 5 µL Mg2+-ATP (final concentration, 100 µm); (ii) 2 µL PKA solution (final concentration, 20 U enzyme/mL in the cis chamber solution); (iii) 5 µL Mg2+-ATP (final concentration, 100 µm) and 2 µL PKA solution (final concentration, 20 U enzyme/mL in the cis chamber solution), followed by dialyzing the cis solution with fresh buffer. In each case, current traces were recorded at clamping potentials of −50 to +50 mV after 30 min of incubation.
Addition of Bax and tBid
After single-channel recording of VDAC was performed, the solution in the cis chamber was removed and fresh buffer was added. Full-length monomeric Bax protein was added to the cis chamber and stirred for 5 min. The concentration of Bax was varied from 10 to 100 nm. After an interval of 30 min, current traces were recorded for 3 h. Then tBid was added to the same chamber in the ratio of 1 : 20 for tBid : Bax, as in our earlier report (Banerjee and Ghosh 2004a), under constant stirring (for 5 min); a recording of current traces was performed after incubation for 30 min. For the control experiment, only tBid and Bax were added to the cis chamber and current recording was performed. In a different set of experiments, the same concentrations of Bax and tBid were added to the cis chamber after phosphorylation of VDAC by PKA in the presence of Mg2+-ATP. To confirm that PKA is phosphorylating VDAC in the presence of ATP, alkaline phosphatase (a non-specific exogenous protein phosphatase) solution was added to the cis chamber to a final concentration of 5 U enzyme/mL. Then current traces were recorded at applied potentials of −50 to +50 mV.
Translocation of cytochrome c
After single-channel recording for VDAC was performed, cytochrome c (Calbiochem, VWR Deutschland GMBH, Darmstadt, Germany) was added to the cis chamber. Voltage was kept constant at +25 mV. After 30 min, the solution from the trans chamber was taken out and its optical density (OD) was measured at a wavelength of 550 nm. Buffer containing 500 mm KCl, 5 mm MgCl2 and 10 mm HEPES (pH 7.4) was used as standard for calibration during OD measurements.
In a different set of experiments, Bax and tBid were added to the cis chamber after reconstitution of VDAC in a planar lipid bilayer. After recording the increase in the channel current caused by the interaction of Bax and tBid with VDAC, solution from the trans chamber was taken out and replaced by fresh buffer simultaneously. The solution removed from the trans chamber was used as a standard for calibration during the spectroscopic analysis at a wavelength of 550 nm. Cytochome c was added to the cis chamber at a final concentration of 500 µg/mL. The applied voltage was kept constant at +25 mV. After 5 min, 200 µL of solution was taken out from the trans chamber. It was then diluted five times with the BLM buffer. Then the OD of the solution was measured at a wavelength of 550 nm. Similarly, OD was measured after 10, 15, 20 and 30 min, respectively. Beyond 30 min, the bilayer membrane was damaged.
Phosphorylation of the voltage-dependent anion channel–Bax–tBid complex
As soon as there was an increase in the channel current of VDAC, as compared to its native state, after addition of Bax and tBid in the above-mentioned amount, the following additions were made to the cis chamber: (i) Mg2+-ATP to a final concentration of 100 µm; (ii) Mg2+-ATP to a final concentration of 100 µm and PKA solution to a final concentration of 40 U enzyme/mL in the cis chamber solution, followed by dialyzing the cis solution with fresh buffer. In each case, current traces were recorded at clamping potentials of −50 to +50 mV after 1 h of incubation.
Dephosphorylation of the complex
To confirm that PKA was phosphorylating the complex in the presence of ATP, alkaline phosphatase (a non-specific exogenous protein phosphatase) solution, at a final concentration of 5 U enzyme/mL, was added to the cis chamber. Then current traces were recorded at applied potentials of −50 to +50 mV.
Analysis of electrophysiological data
Steady state conductance (current/voltage) of VDAC was calculated from the single-channel current data using the software Clampfit (pClamp 9.0; Axon Instruments). Similarly, conductance was measured after phosphorylation of VDAC, addition of Bax and tBid, phosphorylation of the VDAC–Bax–tBid complex, and the alkaline phosphatase-treatment of the complex. Finally, a comparison was made for the conductance values of native VDAC, phosphorylated VDAC, the VDAC–Bax–tBid complex, the phosphorylated VDAC–Bax–tBid complex and the alkaline phosphatase-treated complex.
Results and discussion
In order to achieve the above-mentioned goal, we have undertaken the following in vitro studies in bilayer electrophysiology. In step 1, purified rat brain mitochondrial VDAC (as revealed by the sodium dodecyl sulphate–polyacrylamide gel electrophoresis profile shown in Fig. 1), when reconstituted in a planar lipid membrane, showed voltage-dependent gating (Fig. 2a). When only ATP (but not PKA) or only the catalytic subunit of PKA (but not ATP) was added to the cis chamber, no change in the current or conductance was observed. However, when both ATP and the catalytic subunit of PKA were added to the cis chamber, a large decrease was observed in the steady-state single-channel current and conductance of VDAC measured at all voltages (−50 to +50 mV), as shown in Figs 2(f) and 3(a). This indicates that phosphorylation of VDAC leads to closing of the channel. These results are different from an earlier report, where phosphorylation of rat liver VDAC showed closure only at negative clamping potential (Bera and Ghosh 2001). Although mitochondrial VDAC purified from various sources showed highly conserved characteristics, such as specific single-channel conductance, voltage-gating parameters and ion selectivity, there was a considerable difference as far as the biochemical characteristics were concerned, for example, amino acid sequence of the sensor region (Rostovtseva et al. 2005). That is probably why the effect of the modulation of rat brain VDAC as a result of phosphorylation by PKA was different from that of rat liver VDAC.
Also, we found that, after application of alkaline phosphatase, VDAC regained its native conductance (Fig. 2g). This showed that VDAC undergoes reversible phosphorylation in the presence of PKA and ATP.
In step 2, after reconstitution of VDAC in planar lipid membrane, full-length monomeric Bax was added to the chamber, but no change in the single-channel current of VDAC was observed, even 3 h after the addition of Bax. The concentration of Bax was gradually varied (from 10 nm to 100 nm), but still no change in the VDAC conductance was observed (Fig. 2b). The experiment was repeated at various potentials, but the observations were the same. After addition of tBid to the same chamber, a significant increase in the current was observed (Fig. 2c). As in our earlier report (Banerjee and Ghosh 2004a), analysis of the current recordings revealed that there is no significant difference in the conductance values of VDAC before and after addition of Bax. Thus, interaction of VDAC and Bax takes place without changing the gating of the channel. This is supported by our earlier finding (Banerjee and Ghosh 2005) that the power spectrum of the native VDAC, which follows power law and 1/f noise pattern, shows white noise after addition of Bax, indicating interaction. However, after addition of tBid, there is a large increase in the channel conductance, as shown in Fig. 3(b). Although it is known that tBid triggers channel formation by Bax (Roucou et al. 2002), we believe that the increase in channel current is not a result of the separate channels formed by tBid and Bax. The reasons are as follows. In earlier reports, the conductance of channel formed by Bax in planar lipid bilayers was 5.6 pS at neutral pH (Shimizu et al. 2000), which is far less than the increase in conductance reported in our results (3–12 nS). Also, it is known that tBid itself forms a channel in planar lipid bilayers, but cardiolipin is necessary for the incorporation of tBid into the bilayer (Rostovtseva et al. 2004). Here, to avoid the insertion of tBid into the membrane and the formation of a channel, we have not used cardiolipin in the formation of the bilayer. In our experiments, we observed that when Bax and tBid were applied to the planar lipid bilayer a channel with very low conductance was formed (Fig. 2i). Therefore, the increase in channel conductance could only be a result of the increase in the pore size of VDAC after its interaction with Bax and tBid. In a different set of experiments, when single-channel VDAC recording was performed, cytochrome c was added to the cis chamber. We found that there was no change in the absorbance of the solution in the trans chamber with native VDAC after 30 min (OD550 = 0.004). This indicates that translocation of cytochrome c does not occur when only VDAC (native) is present in the bilayer. Also, no change in the absorbance was observed when Bax was added (OD550 = 0.005). But when cytochrome c was added to the cis chamber, after a Bax–tBid-mediated increase in pore size of VDAC was observed, there was a gradual increase in the absorbance of trans chamber solution (as shown in Table 1). This indicates that translocation of cytochrome c from the cis chamber to the trans chamber does not occur when only VDAC (native) is present in the bilayer. But there is movement of cytochrome c from the cis to the trans chamber when the VDAC–Bax–tBid complex is present in the bilayer membrane.
Table 1. Translocation of cytochrome c: absorbance of the trans chamber solution at 550 nm after Bax–tBid-mediated increase in the pore size of the voltage-dependent anion channel (VDAC) at various time intervals
OD550 of trans chamber solution
Data shown is ± SD of three independent experiments.
0.409 ± 0.007
0.453 ± 0.009
0.492 ± 0.005
0.52 ± 0.007
In step 3, Bax and tBid were added to the cis chamber after a decrease in the channel current as a result of phosphorylation of VDAC by PKA. We observed that there was no change in the channel current (Fig. 2h). In other words, the increase in conductance as observed in the case of interaction of native VDAC with Bax and tBid was not seen in the case of phosphorylated VDAC.
In step 4, once the increase in VDAC conductance was recorded after addition of Bax and tBid, ATP (but not PKA) was added to the cis chamber, but no change in the current or conductance was observed, which means that the conductance of the VDAC–Bax–tBid complex still remained the same. But, when the catalytic subunit of PKA along with ATP was added to the cis chamber, it showed a large decrease in steady state channel current measured at all voltages (−50 to +50 mV), as shown in Figs 2(d) and 3(b). Thus, after phosphorylation of the VDAC–Bax–tBid complex with PKA, there was a closure of the channel, as observed from the voltage versus conductance plot (Fig. 3b). The channel conductance progressively declined and after 1 h the channel conductance achieved a residual state of conductance of 0.33–0.6 nS. The amount of PKA required to phosphorylate VDAC after its interaction with Bax and tBid is double the amount required to phosphorylate native VDAC. A plausible reason for a higher (double) critical concentration is that 20 U is enough to modulate native VDAC, but after addition of Bax and tBid there is a huge increase in the pore size of VDAC, which is expected to expose additional serine residues (potential phosphorylation site) buried inside the native protein (other than those exposed in the native conformation), hence the increased demand for the PKA required to phosphorylate all these serine residues.
In the last step, to check the reversibility of the phosphorylation of the complex, alkaline phosphatase was added to the same chamber. This showed a significant increase in the channel conductance (almost equivalent to the native VDAC channel) of the complex at all the applied potentials (−50 to +50 mV). Figure 1(f) shows the current–time trace of the VDAC–Bax–tBid complex at +25 mV after the addition of alkaline phosphatase.
Phosphorylation of the VDAC–Bax–tBid complex by PKA leads to the closure of the channel, similar to the action of PKA on native VDAC. Interestingly the conductances in both native and Bax–tBid-bound VDAC after phosphorylation are of the same order. We speculate that this happens because of structural modifications in VDAC caused by phosphorylation (not of Bax or tBid). Accordingly, the Bax and tBid proteins bound to VDAC either do not occupy the phosphorylation sites or are dissociated. So, when alkaline phosphatase was added we observed that the conductance value equivalent to native VDAC was regained (Figs 2d and f).
The in vitro results discussed above highlight the importance of phosphorylation in resisting leakage of cytochrome c through VDAC. If true in in vivo conditions, this finding brings a new understanding of combating apoptosis by cells. Especially in the brain, although normal aging is characterized by modest reductions in the mass and volume of the human brain, which is caused by the death of brain cells, these changes are far more profound in patients who succumb to neurodegenerative disorders like Alzheimer's disease (MacGibbon et al. 1997; Su et al. 1997) and Parkinson's disease (Vila et al. 2001). Given the fact that there is no single cause or pathologic mechanism for these disorders, cytochrome c-induced apoptosis is one of the major causes for brain cell death (Clayton et al. 2005). As VDAC is the channel protein responsible for the release of cytochrome c during the apoptotic condition, regulation of this channel has a significant role in the control of cell death. We believe that cells resist apoptosis by controlling transport through VDAC by means of phosphorylation. Similar results for the inhibition of cytochrome c release through mitochondrial VDAC channels by C-Raf kinase was also reported (Le Mellay et al. 2002). Obviously, the cell must have a mechanism to activate kinases for this purpose.
Decoding of this mechanism of control of cell death will throw light on the role of apoptosis in normal nervous system development and also in the cases of neurodegenerative disorders. The enormous number of people who suffer from ailments that range from Alzheimer's disease to Parkinson's disease may see some relief if this regulation is implemented to preserve cells by interrupting the cell death messages.
We would like to give our sincere thanks to Professor Jean-Claude Martinou, Department de Biologie Cellulaire Sciences, Ernest-Ansermet, Geneva, Switzerland for providing us with the recombinant full-length monomeric Bax and tBid. We also thankfully acknowledge the Department of Science and Technology (Government of India) and Council of Scientific and Industrial Research (Government of India) for financial support.