Mitochondrial Outer Membrane Channels - ACS Publications

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Mitochondrial Outer Membrane Channels Marco Colombini* Department of Biology, University of Maryland, College Park, Maryland 20742, United States by the rest of the cell and vice versa. This mutual regulation occurs at various levels, including the control of the flux of soluble components. Membrane channels are the major conduits of matter across the outer membrane. Their regulation is thus critical to the control of the flow of matter. This “matter” includes ions, metabolites, and intact proteins. The most important inorganic ions for mitochondrial function are H+, phosphate, and Ca2+. Among the myriad of metabolites, perhaps the most important are pyruvate, ADP, ATP, creatine, and phosphocreatine. Voltage-dependent anion channels (VDACs) are the pathways by which small ions and metabolites cross the outer membrane,1,2 and therefore one might expect that these channels are particularly adapted to the translocation of these small molecules. This adaptation would CONTENTS include both facilitating and controlling the translocation 1. Introduction A process. The gating of VDAC controls the flux of these 2. VDAC Channels: Overview B substances in complex ways, some of which have been 2.1. VDAC Structure B elucidated. In addition to small molecules, proteins must be 2.2. VDAC Regulation by Voltage Gating C able to translocate between the cytosol and mitochondrial 2.3. VDAC Regulation and Mitochondrial Funccompartments. Protein import is critical because most tion E mitochondrial proteins are encoded by the nuclear genome 2.4. Consequences of the Lack of an Individual and produced in the cytosol. The import of these proteins is VDAC Isoform F selectively controlled by the translocator of the outer 2.5. VDAC’s Role in Apoptosis F mitochondrial membrane (TOM).3 The need to select which 3. Protein-Release Pathways in the Outer Memcytosolic proteins are imported into mitochondria and to move brane F them in a unidirectional manner requires an import machinery, 4. Ceramide Channels as a Protein-Release Pathway G not a free-flowing channel. Although, when reconstituted into 4.1. Ceramide Permeabilization of the Mitochonplanar membranes, TOM forms channels,4 clearly the process is drial Outer Membrane H more complex and not the topic of this review. The protein 4.2. Structure of Ceramide Channels H translocation process performed by outer membrane channels 4.3. Gating of Ceramide Channels H is the rapid release of intermembrane space proteins to the 4.4. Regulation of Ceramide Channels I cytosol. The channels capable of freely translocating proteins 4.5. Ceramide Channels and Disease J across the outer membrane form in the early stages of apoptosis 5. Channel Formation by Bax and Bak J because protein release from mitochondria activates the 5.1. Visualization of Bax Channels J execution phase of apoptosis. 5.2. Bax Channel Formation in Phospholipid It is remarkable that the same organelle that is so critical to Membranes J the function of the eukaryotic cell also has the task of deciding 5.3. Mitochondrial Apoptosis-Induced Channel K whether the cell should continue or be recycled. Apoptosis, the 5.4. An Integrated View of Bax/Bak Channels? L conversion of a cell into food, takes place when a cell is 6. Conclusion and Perspectives L damaged or no longer needed. Apoptosis plays a critical role in Author Information L all the major diseases of developed countries where Corresponding Author L malnutrition and infectious diseases have been greatly reduced Notes L if not eradicated. Stroke, heart disease, cancer, viral diseases, Biography L and neurodegenerative diseases all involve problems with the Acknowledgments M apoptotic process. Cell death from inappropriate initiation of References M apoptosis is a major problem in stroke, myocardial infarction, and neurodegenerative diseases. Lack of apoptosis is a major

1. INTRODUCTION The outer membrane of mitochondria is at the interface between the cytosol and the mitochondrial environment. It is therefore the ideal site for regulation of mitochondrial function © XXXX American Chemical Society

Special Issue: 2012 Ion Channels and Disease Received: May 22, 2012

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2.1. VDAC Structure

problem in cancer. Viral inhibition of apoptosis in the infected cell allows viruses to replicate and spread. Control of mitochondria-mediated apoptosis should be an effective way of treating these diseases or minimizing the damage the diseases produce. The mitochondrial intermembrane space contains proteins that when released into the cytosol will activate pro-enzymes of the caspase cascade, leading to the execution phase of apoptosis. Under normal conditions the outer membrane is impermeable to proteins, thus keeping physically separated the cytosolic pro-enzymes from the proteins that would initiate their activation. Therefore the binary weapon whose purpose is to eliminate damaged or unwanted cells is kept inactive by the outer membrane until needed. Thus, the channels in the outer membrane perform different roles and have very different structures and properties.

The VDAC channel is formed by a single polypeptide8,9,16 folded into a largely β-barrel structure.17 The single layer of protein separating the aqueous pore from the hydrocarbon tailrich region of the membrane places strong constraints on the physical properties of the transmembrane strands. These constraints allow one to identify the regions in the protein sequence that have the appropriate characteristics to be transmembrane strands.18 Although the primary sequence does vary rather widely depending on the source species, the hydrophobic/hydrophilic pattern that drives the formation of the channel is highly conserved in VDAC from species as diverse as mammals, fungi, and higher plants.10 Thus, the basic structure of VDAC evolved very early, perhaps concomitant with the formation of the symbiotic relationship between the original mitochondrial ancestor and its host. Some would extend the lineage back to the porin channels that permeabilize the bacterial outer membrane. Regardless of the origin, the fundamental properties of voltage gating, anion selectivity, and single-channel conductance were either defined early and retained by strong selective pressure or are a unique solution to the formation of a channel with the properties and regulation necessary for optimal cellular function. Indeed, these basic properties of the archetypal VDAC channel, found in all mitochondria examined,19 are so close, despite a billion years of evolutionary separation, that one could conclude that altering these properties in a species leads to the extinction of that species. The alternative that point mutations do not alter the properties is not correct because single-point mutations at each of a variety of sites results in marked changes in these properties,20,21 changes not seen in the wild-type protein from many organisms. There are isoforms of VDAC22 whose properties are different,23 but the archetypal form has the same basic properties. The conserved properties, coupled with the conserved pattern of hydrophobic/hydrophilic amino acid residues, argue for a common channel structure. The basic structure of the VDAC channel is a topic of some controversy among current investigators. The solving of essentially the same 3D structure24−26 by NMR and X-ray crystallography has convinced many that this is the structure of the native VDAC channel. This 19-stranded β-barrel with the N-terminal α-helix intruding into the pore is very attractive from a structural point of view. All the regions on the primary sequence that have a hydrophobic/hydrophilic pattern consistent with forming the inner wall of the channel are included in the 19 beta strands.27 The problem is that this beautiful structure is in conflict with many experiments performed on VDAC for many years.27 These contradictions are conflicts with hard data and thus are not easily dismissed. Indeed, three independent lines of investigation, performed on VDAC channels reconstituted into planar phospholipid membranes, provide compelling evidence for the regions of the VDAC protein that form the transmembrane strands. Selectivity changes resulting from point mutation introduced charge changes at specific sites determined whether a putative transmembrane strand was indeed transmembrane.10,21 This was done for both the open and closed states (recall that the closed state is still permeable to small ions) and thus not only identified the transmembrane strands in the open state but those that moved out of the channel upon channel closure. The second line of investigation20 was the quantitation of changes in the gating charge using the same charge-substituted point mutations (see section 2.2 for more detail). The third28 was to

2. VDAC CHANNELS: OVERVIEW The selective translocation of metabolites by a variety of carriers occurs at the inner membrane. The regulation at the outer membrane is, by necessity, a far coarser process because it uses a common pathway, the VDAC channel.2 However, VDAC’s role as a common pathway makes this channel very important because its malfunction affects many systems. In addition, VDAC’s mode of action must reflect a compromise among the functional requirements of competing processes. The complex regulation that executes this compromise acts on a remarkably small protein that is capable of responding to a variety of physical and chemical signals. VDAC is a 30 kDa,5−7 monomeric,8,9 and highly conserved protein.10 It is regulatable in a number of ways; only some of these are clearly linked to a physiological function. Clearly, properties that are highly conserved evolutionarily must have a physiological significance even if such has not yet been discovered. Therefore, this part of the review will explore the various properties of this remarkable and versatile channel and the deleterious effects of knocking out even a single VDAC isoform. VDAC’s critical role in metabolite transport and cellular energy production makes it an attractive target for the development of anticancer drugs.11 As efforts are underway to identify specific inhibitors or modulators, it is interesting to note that active compounds, either designed for a different purpose or identified in a screen for genotype-specific lethality, turned out to act by targeting VDAC. Both efforts were aimed at killing cancer cells in a specific manner. The first involved the generation of an oligonucleotide that was antisense to the initiation codon region of Bcl-2 mRNA.12 To stabilize the oligonucleotide, a phosphorothioate oligonucleotide was generated. It does silence the Bcl-2 mRNA and reduce the expression of the Bcl-2 protein. This should reduce the resistance of tumor cells overexpressing Bcl-2 to chemotherapy agents. However, by using the phosphorothioate version, the researchers inadvertently made a compound that contributes to the killing of tumor cells not by silencing the Bcl-2 mRNA but by blocking VDAC,13 among other actions. The second effort was a large-scale screening of compounds that would kill tumor cells harboring the oncogenic H-Ras and not normal cells.14 The active compound, called erastin, turned out to act on VDAC.15 These examples show how disturbing the function of VDAC can result in significant deleterious effects on cells. B

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introduce cysteine residues, biotinylate them, and then identify their surface location based on accessibility to streptavidin and the influence of streptavidin binding on the state of the channel. The experimental data are consistent with a channel formed by 1 α-helix and 13 beta strands27,28 (Figure 1, bottom). It is

Figure 2. VDAC crystals in the mitochondrial outer membrane of Neurospora crassa. (A) Electron micrograph of an outer membrane vesicle that has been freeze-dried and shadowed with metal. The twodimensional crystal takes up most of the surface of the vesicle, forming a very flat region consisting of raised bumps in an ordered array. (B) Optical diffraction pattern of a VDAC crystal in an outer membrane vesicle. (C) Topology of VDAC channels obtained from the analysis of VDAC crystals. The raised region in the middle of the six-channel repeating crystallographic unit is likely formed by the surface domains of the VDAC channels. (D) Averaged image of an electron micrograph of a negatively stained VDAC crystal. The stain is filling the pores formed by the array of VDAC channels. A thorough study that was the source of the images was published in ref 8.

Figure 1. Folding patters of human VDAC1 determined by NMR24 (3-D) and deduced from functional studies33 (functional). The large red boxes indicate the transmembrane beta strands found in the 3-D structure that are proposed to be located in surface domains in the functional structure. The residues boxed in red are sites tested by mutagenesis where the charge at the site was altered and there was no effect on channel selectivity.10,21,28,32,33 The mutations were made on VDAC from S. cerevisiae 21 and human VDAC1, 10 and the corresponding locations are indicated in the figure. The green numbers indicate the location of the insertion of the FLAG epitope in studies aimed at defining the topology of VDAC in yeast mitochondria.29 Sites 2 and 3 face the intermembrane space whereas sites 4 and 5 face the cytosol. The numbers next to the amino acid residues are the amino acid number starting at the N-terminus (Nterminal methionine removed). The circled residues indicate the ends of the transmembrane beta strands.

channel were obtained.8 The folding pattern of the channel is drawn to scale in Figure 3. This structure can be reconciled with the NMR/X-ray structure if one considers that the protein used in the latter studies was refolded in vitro from denatured protein and can be converted to the functional protein by a rather mild treatment.24 A reasonable structural change was proposed that could convert the NMR/X-ray structure into the functional structure.27 A detailed analysis of this controversy based on experimental evidence was published recently,30 concluding that the 3D structure is not the structure of the voltage-gated channel reconstituted into phospholipid membranes nor the channel that exists in mitochondria. An alternative opinion was published that largely ignored the experimental evidence inconsistent with the 3D structure.31 2.2. VDAC Regulation by Voltage Gating

interesting to note that all the transmembrane beta strands identified by the functional studies were included in the 3D structure (Figure 1 top). In the remaining strands (Figure 1, large red boxes), charge substitutions at sites boxed in red had no effect on channel ion selectivity, demonstrating that these charged residues were far from the channel lumen10,21,27 and thus the associated strand could not be transmembrane. The topology of VDAC was explored29 in yeast mitochondria by inserting the FLAG epitope at key locations indicated in Figure 1 by the green numbers 2−5. Locations 2 and 3 were found to face the intermembrane space whereas 4 and 5 were cytosolic, in agreement with the folding pattern deduced from functional studies (Figure 1, bottom) and in disagreement with the 3-D structure (top). Figure 2 shows a 2-dimensional crystal of VDAC channels formed in the outer membrane isolated from N. crassa mitochondria. By using Fourier transform-based filtration and averaging, low-resolution images of the topography of the

Voltage gating requires the movement of charge through the transmembrane electric field. Thus, some region of the protein must be capable of motion, having a low energy barrier to such motion. This requires a weaker interaction with the rest of the protein. In addition the motion must be coupled to a change in the ability of the protein to conduct the important solutes that are the most critical function of the channel. Point mutations that altered the charge of specific regions of the channel were engineered into VDAC to identify the regions that are involved in this domain motion.20,32,33 This domain would be the voltage sensor. The regions identified were the α-helix and adjacent beta strands: the C-terminal beta strand and 4 beta strands in the N-terminus. It is fitting that the motion should take place at the site of the unappealing interloper, the α-helix imbedded into a β-barrel. This is likely to be the weakest point in the structure and thus have the lowest energy barrier for motion. At all except one site,20 the change in the steepness of C

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nm to a measured37 1.8 nm (from the ability of gamma cyclodextrin to barely permeate). This steric constraint contributes but does not explain the fact that the open state of VDAC allows the flow of ATP whereas the “closed” state does not.38 (Closed refers to the lower conductance states resulting from voltage gating.) The major change is a radical change in the net charge on the wall of the channel resulting from the extrusion of the positively charged domain.32,33 Thus, the channel goes from being anion-preferring to being cationpreferring, as measured by the selectivity to small ions.39−41 However, the story is even more interesting in that a set of mutations that cause essentially the same change in selectivity for small ions as the closing process does not have much effect on the ability of ATP to cross the membrane.42 This demonstrates that ATP translocation requires interaction with specific sites on the channel wall that interact with ATP in a complex and complementary way. Indeed, molecules of the same size and charge as ATP can be excluded by VDAC’s open state.43 Upon channel closure, the movement of the voltage sensor must disrupt this hand-in-glove fit and likely actually introduce unfavorable interactions so that the channel, despite being quite a bit larger than the size of ATP, actually excludes ATP. Clearly these finding provide compelling evidence that the voltage-gating of VDAC is evolutionarily selected and maintained to control the flow of ATP/ADP between mitochondria and cytosol. VDAC is not a general diffusion pore but a highly tuned molecular machine. The large motion of the protein proposed for the gating process is sometimes greeted with skepticism with worries of the possibly large activation energy associated with this structural change. Intuition is sometimes a poor guide but, in any event, must be guided by experimental findings. The large motion, bringing to the membrane surface areas that face the pore lumen in the open state, makes two clear predictions: (1) charged residues in this mobile region should influence the selectivity of the channel in the open state but not in the closed state; (2) in the open state such regions would not be able to bind to soluble proteins but binding may occur upon channel closure. Indeed such binding should block channel reopening. Both of these expectations were found to be true.32,33 Other consequences of this gating mechanism are as follows: a large change in pore volume upon channel closure, and voltagedependent closing rates and voltage-independent opening rates. These were also observed experimentally.44,30 Depending on the molecular details, one might expect that the packing of VDAC channels into 2-dimensional crystals would be different if the channels were in the open or closed state. However, no significant change in packing was observed.45 If voltage gating were important physiologically, one would expect that a voltage would be present across the mitochondrial outer membrane and that this voltage would change with changes in the state of the cell. It is rather common to assume that there cannot be a potential across the outer membrane because of the large permeability of VDAC channels in both their open and closed conformation. Gradients of small ions would surely reach electrochemical equilibrium. However, electrochemical equilibrium does not equate to zero potential. Both the cytosol and the mitochondrial intermembrane space are filled with charge polymers, such as proteins and nucleic acids. These polymers cannot equilibrate across the outer membrane. Unless the charge of these membrane-impermeable polymers is identical in both compartments, there must be a Donnan equilibrium potential across the outer membrane.

Figure 3. Structure of the VDAC channel as deduced from many experiments. This differs from the crystal and NMR structure, which is likely not the native structure. The wall of the channel is formed by one α0helix (ovoid in panel A) and 13 beta strands (trapezoids in panel A). The transmembrane strands tilted at 46 degrees are shown in panel B. Because of the tilt, regions were defined by the dotted lines (B), and net charge in those regions is indicated by the colors in paenl A (red for negative and blue for positive). An ATP molecule was inserted in a likely preferred orientation. Reprinted with permission from ref 30. Copyright 2012 Elsevier B.V.

the voltage dependence caused by the engineered charge change was half that of the engineered change, indicating that the site moves only halfway through the membrane. At the remaining site, the values were the same, indicating complete translocation. These sites were on the channel inner wall and thus located in the electric field. The movement of the strands from being transmembrane to the membrane surface would cause the sites tested to move roughly halfway through the electric field. Increasing the positive charge increased the steepness of the voltage dependence, whereas introducing a negative charge reduced the steepness. The calculated difference in conformational energy between the states did not change by very much, supporting the conclusion that the mutation resulted in little if any structural change. It is interesting to note that converting amino groups in VDAC to carboxyl groups by succinylation34 results in loss of voltage dependence at low doses but restores it at high doses, indicating that any net charge in the voltage sensor region allows the region to be driven out of the channel upon the application of an electric field. The mechanism just described automatically couples the gating process to major changes in the permeability properties of VDAC. Reducing the perimeter of the channel by 5 or 6 strands reduces the pore diameter from an estimated35,36 2.5 D

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tubulin dimers. Tubulin was found to block VDAC channels reconstituted into phospholipid membranes by inserting its negatively charged tail into the channel.56,57 This blockage mimics the drop in conductance and inversion of selectivity seen with VDAC voltage gating, indicating that in both cases it is beneficial to maintain the flow of small ions while inhibiting the flux of ATP and other anionic metabolites. The dynamic block by tubulin would link mitochondrial function to cellular activity. Tubulin polymerization occurs when cellular activity increases, resulting in a reduction in the concentration of tubulin dimers and thus a reduction in VDAC blockage. Indeed, microtubule depolymerization using colchicine results in a reduction in inner mitochondrial membrane potential whereas the opposite was observed when microtubule polymerization was enhanced.58 In a similar way, G-actin, but not F-actin, was found to increase the voltage-gating of VDAC, and the formation of F-actin is associated with increased cellular activity.59 Both effects depend on voltage because tubulin blockage has a steep voltage dependence, equivalent to the translocation of 10 elementary units of charge through the membrane. The high voltage dependence would only make VDAC closure or blockage more sensitive to the transmembrane potential and thus more responsive to other processes that affect the potential. Another level of “potential” regulation and thus complexity is that tubulin blockage is sensitive to the degree of VDAC phosphorylation.60 The exposure of liver cells to ethanol leads to rapid changes in the metabolic state of cells, called “swift increase of alcohol metabolism” (SIAM). These changes aid in ethanol and acetaldehyde detoxification but may also lead to fatty liver or steatosis. Strong evidence61 points to VDAC closure as a leading factor in the onset if SIAM. For example, ethanol exposure reduces the ability of plasma membrane permeabilized hepatocytes to undergo succinate-dependent respiration, but outer-membrane damage restores the respiration. VDAC closure blocks the flow of normal metabolites such as pyruvate and fatty acyl-CoA but not the flow of the lipid-soluble acetaldehyde. The matrix aldehyde dehydrogenase converts the acetaldehyde to acetate. The uncoupling that also occurs under these conditions allows acetate to be further consumed without the need for ADP to store the energy thus produced and normally transferred to the protonmotive force. The preferential metabolism of acetaldehyde leads to rapid detoxification at the cost of cytosolic ATP depletion, inhibition of ureagenesis, and fat deposition. In hepatoma cells, tubulin blockage of VDAC reduces mitochondrial membrane potential and contributes to the suppression of mitochondrial metabolism, perhaps contributing to the Warburg effect, i.e., cells rely on glycolysis even when oxygen is available for oxidative phosphorylation. There is a case where evidence points to the role of VDAC, but the mechanism is still uncertain.62 In ischemia/reperfusion injury of the heart, the anoxic period results in mitochondrial depolarization, leading to a reduction in cellular ATP, leading to an increase in glycolytic rate, leading to acidification of the cytosol surrounding the ischemic region, leading to an increase in cytosolic Na+ levels, leading to elevation in cytosolic Ca2+ . Upon reperfusion and reenergization, restoration of the mitochondrial potential is associated with a burst of ROS (reactive oxygen species) and Ca2+ accumulation into the mitochondrial matrix. Both elevate the risk of mitochondrial permeability transition and cell death by a combination of necrosis and apoptosis. Short ischemic episodes render the

Measurements of the delta pH across this membrane also measure the membrane potential difference, assuming that the protons have equilibrated thanks to the high permeability of the VDAC channels. Such measurements have been published by two independent groups,46,47 both showing that the intermembrane space is negative compared to the cytosol. The two reported measurements are different (one corresponds to −20 to −30 mV and the other to −40 mV), perhaps indicating that the potential can indeed change under different conditions. Furthermore, metabolism does result in the flux of charged species across the outer membrane, and different molecular forms move in different directions. For example, there is a net movement of ADP into the mitochondrion whereas there is a net movement of ATP out of such. These have different charges and thus different influences on the potential across the outer membrane. Theoretical calculations of the results of such dynamic fluxes indicate that significant potentials would be generated,48 and these depend on metabolic rates. In a simple case, high metabolic rates would generate higher potentials that would favor VDAC closure and vice versa. This would act like the governor of a steam engine, as an automatic negative feedback process. 2.3. VDAC Regulation and Mitochondrial Function

The gating of VDAC, whether by voltage gating or by other regulatory processes, obviously changes the ability of molecules and ions to cross the outer membrane but does so in surprising ways. As stated above, the flux of anionic metabolites is greatly reduced or eliminated by a combination of a reduction in channel size (steric barrier) and an inversion in the net charge and charge distribution within the channel (electrostatic barrier). However, for the important ion Ca2+, channel closure actually increases its permeability49 because the electrostatic effects are more pronounced than the steric effects. In addition, there are a variety of “closed” states with different properties,36,50 and thus it is possible that different modulating agents may alter the permeability properties of VDAC in different ways to achieve different permeability characteristics. Indeed, modulating factors do seem to favor particular closed conformations. Perhaps the first clear demonstration of a physiological function to VDAC gating was the observation of failure to exchange ATP/ADP across the mitochondrial membrane in mammalian cells deprived of growth factor.51−53 Prior to the release of proteins leading to end-state apoptosis, growth factor removal results in VDAC closure and the accumulation of phosphocreatine in the intermembrane space. Creatine is a low molecular weight zwitterion, lacking net charge, and thus would be expected to permeate through both the open and closed states of VDAC. Its phosphorylation by creatine kinase in the intermembrane space converts it into a doubly charged negative ion, expected to be unable to translocate through the closed state of VDAC. Experimental results confirmed this expectation,53 accounting for the large accumulation of phosphocreatine 9 h after growth factor withdrawal. This closure of VDAC is proposed to be part of the signaling system that leads to the release of proteins from mitochondria. Muscle cells with permeabilized plasma membranes were used to study mitochondrial function in situ, and it was found that the apparent Km for ADP-dependent respiration was much higher than expected and could be reduced by an order of magnitude by damaging the outer membrane.54,55 A soluble cytosolic factor was responsible, and this turned out to be E

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lack of sperm motility. The distribution of mitochondria in neurons was abnormal,71 consistent with difficulty in delivering mitochondria to the nerve terminal. The knockout of VDAC2 in mice is embryonically lethal.72 This is thought to be associated with the binding of VDAC2 to BAK. Conditional deletion of VDAC2 in lymphocytes results in rapid cell death, and this is prevented by the concomitant deletion of BAK.73 A conditional VDAC2 knockout in the heart results in progressive fibrosis and cardiomyopathy.67 Knockouts of each or both of the other two isoforms produces viable offsprings with interesting defects. VDAC3 knockout males are sterile due to very low sperm motility.74 The axoneme in these sperm is missing a microtubule doublet due to a defective maturation process. VDAC3 expression is particularly plentiful in gonads, and the lack of VDAC3 somehow influences the structure of the axoneme. VDAC1 knockout results in mitochondrial hypertrophy and reduction in ADP-dependent respiration.75 The generation of more and larger mitochondria is typical of diseases on mitochondrial insufficiency, but in this case, the problem is metabolite exchange across the outer membrane. A similar finding was observed for the VDAC3 knockout, but this was limited to cardiac tissue.76 The VDAC1 knockouts were also glucose-intolerant in a manner similar to adult-onset diabetes.67 The overarching message from the knockout experiments is that VDAC isoforms have distinct functions, and these functions go well beyond what one would expect from channel formation.

heart resistant to permanent damage by cell death. Similarly, overexpression of Bcl-2 is also protective. The hypothesis is that VDAC closure would be protective in preventing glycolytic ATP from being consumed by the mitochondrial proton ATPase under depolarizing conditions. It is controversial whether antiapototic proteins favor VDAC opening or closure. From experiments with pure proteins, Bcl-xL shifts the switching region favoring channel opening,52 but in vivo, the conditions may be such that that the effect is reversed due to a combination of factors acting on VDAC. An example of a cellular process in which the proposed involvement of VDAC turned out to be incorrect is the mitochondrial permeability transition. VDAC, the adenine nucleotide translocator, and the cyclophilin D were proposed to form a complex that spanned both mitochondrial membranes and was capable of resulting in translocation of small molecules between the cytosol and the matrix compartment under appropriate conditions. Now it seems clear that the permeability transition is strictly an inner-membrane phenomenon. The permeability transition takes place even when any of the VDAC isoforms are knocked out.63 For a thorough review, see ref 64. Undoubtedly, more examples of VDAC regulation of cellular processes will come to light now that the role of VDAC is included in the hypotheses of investigators. Unfortunately, one often only finds what one looks for. 2.4. Consequences of the Lack of an Individual VDAC Isoform

2.5. VDAC’s Role in Apoptosis

Although the mitochondrial channels most intimately involved in apoptosis are those that allow proteins to cross the outer membrane, VDAC also plays a role in the process as already mentioned in the previous two sections. Of the three VDAC isoforms identified in mammals, VDAC2 binds to the proapoptotic protein, BAK.72 In doing so it can, at times, inhibit the apoptotic process by sequestering BAK, making it unavailable to form channels.72 In other situations VDAC2 is needed to recruit BAK to the mitochondrion77 so that BAK may form channels or contribute to channel formation. The role of VDAC2 in favoring apoptosis has been reported as critical to providing resistance to infectious bursal disease in avians.78 By favoring apoptosis during viral infection, VDAC2 slows down viral replication.

In mammals there are 3 VDAC isoforms, labeled VDAC1, ̈ notion that the existence VDAC2, and VDAC3.65,66 The naive of 3 VDAC isoforms is solely to provide some redundancy to ensure that molecular trafficking across the outer membrane continues under different conditions is totally wrong. Moreover the existence of several alternatively spliced versions indicates the need for an even greater variety of properties. Knockout studies in yeast, drosophila, and mice reveal unexpected and dramatic changes in cellular and whole-organism properties.67 Clearly just one form of VDAC is insufficient to satisfy all the metabolic requirements of cells, especially in complex multicellular organisms. The yeast, S. cereviciae, has 2 VDAC isoforms.68 VDAC1 is the canonical VDAC with the conserved properties. VDAC2 is either unable to form channels or has very low channel-forming ability.69 Isolated yeast mitochondria containing just VDAC2 have a very low permeability to NADH, essentially the same as that of mitochondria lacking both VDAC1 and VDAC2. The baseline level could easily be attributed to the small fraction of damaged mitochondria in the isolated fraction. Yet VDAC2 can compensate for VDAC1 in terms of allowing the yeast to grow under nonpermissive conditions. This indicates the existence of other functions for VDAC over and above providing a pathway for metabolite translocation. Drosophila melanogaster has 4 VDAC isoforms.70 One of these, the canonical form, is ubiquitously expressed. It is often referred to as porin, and for clarity I will call it DVDAC1. When DVDAC1 was knocked out,71 some of the consequences could be expected based on reduced channel function. These were some developmental lethality, altered mitochondrial respiration, and problems with the functioning of nerves and muscles. The consequences at the system and whole-organism levels are less easy to predict. These include abnormal climbing behavior, hypersensitivity to mechanical stress, and male infertility due to

3. PROTEIN-RELEASE PATHWAYS IN THE OUTER MEMBRANE The permeabilization of the mitochondrial outer membrane to proteins results from the formation of large channels. There is disagreement whether swelling of the inner membrane following the formation of the permeability transition results in tearing of the outer membrane,79 and thus the release of intermembrane space proteins into the cytosol, or whether the protein release is controlled by a separate process.80 The swelling of the inner membrane would change dramatically the concentrations of macromolecules in the intermembrane space and, at the same time, increase contact between the two membranes. This may result in the formation of channels in the outer membrane. In addition, the outer membrane can be permeabilized to proteins without swelling of the inner membrane. Indeed, the evidence for the formation of discrete channels that release proteins from mitochondria is overwhelming and not at all controversial. What is controversial is whether the channels formed are well-organized structures or F

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Figure 4. Structure of a ceramide channel. Panels A, B, and D show aspects of the working structural model of a ceramide channel. Panel A shows a 48-column channel forming a 10 nm pore. Panel B shows a smaller channel and how it might interface with the phospholipid bilayer. Panel D shows two antiparallel columns each consisting of 6 ceramide molecules hydrogen-bonded to each other through the amide linkage. The red arrows indicate the directions of the two dipoles formed by the ceramide columns. Panel C shows an electron micrograph of a negatively stained ceramide channel in a phospholipid vesicle (left) and a densitometry tracing of this electron micrograph (right). A detailed study of negatively stained ceramide channels was published in ref 104.

occurs in various membrane compartments, including mitochondria.84−86 Addition of ceramide to isolated mitochondria results in permeabilization of the outer membrane and protein release. 87−89 How this release actually takes place is controversial. That ceramide itself forms the channels is not widely accepted.90,91 Even among those that accept that ceramide forms channels, there are questions regarding the importance of ceramide channels in the release of proteins from mitochondria. The finding that cells lacking both of the main pro-apoptotic proteins, Bak and Bax, are highly resistant to stimuli that trigger apoptosis is sometimes considered as incontrovertible evidence that the release pathway is formed by Bax and/or Bak. However, unlike the parental cells, the Bax/ Bak knockout cells show no increase in mitochondrial ceramide levels upon receiving a pro-apoptotic signal.92 It turns out that Bak is needed for the elevation of ceramide levels through a specific ceramide synthase. Thus, it is not at all easy to exclude the possibility that ceramide channels can serve as the proteinrelease channels. Indeed, simple addition of ceramide to isolated mitochondria results in permeabilization of the outer membrane to proteins.87,89,91−95 This is the case not only for mammalian mitochondria but also for yeast (S. cerevisiae) mitochondria,96 and yeast lack the Bcl-2 family proteins but have ceramide. The regulation of ceramide channels by Bcl-2 family proteins provides strong evidence for the role of ceramide channels in apoptosis.

transient lipidic pores formed by proteins disturbing the lipid bilayer.81,82

4. CERAMIDE CHANNELS AS A PROTEIN-RELEASE PATHWAY Published work provides extensive evidence that a sphingolipid, called ceramide, forms channels in the outer membrane that are one of the protein-release pathways. There is a large and solid literature supporting the conclusion that ceramide is proapoptotic and that elevation of ceramide levels in cells is common early in the apoptotic process and is often essential to the progression of apoptosis.83 For example, a wide variety of factors, both physical and chemical, some added and others withdrawn, that induce apoptosis also result in elevation of cellular levels of ceramide. Blocking this elevation typically blocks apoptosis. The causal relationship between ceramide elevation and apoptosis becomes clear when the manipulation of this level can be used therapeutically. For instance, a wide variety of tumor cells can be induced to undergo apoptosis by using inhibitors of ceramide metabolism so as to artificially elevate ceramide levels or by administering membranepermeable ceramide analogues.83 Resistance to radiationinduced apoptosis can arise from failure of a cell type to elevate ceramide levels in response to the dose of radiation. This elevation of ceramide levels in cells takes place within membranes because, like phospholipids, ceramides formed in cells are essentially insoluble in water. The increase in ceramide G

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4.1. Ceramide Permeabilization of the Mitochondrial Outer Membrane

4.2. Structure of Ceramide Channels

The structure of the ceramide channel (Figure 4) was inferred from functional studies100 and supported by electron microscopic observations104 (Figure 4C), as well as by molecular dynamic simulations.109 Model building initially proposed99 that ceramide molecules (probably 6) would stack, linked by the hydrogen bonding of their amide linkages (in analogy to the linkages in the α-helix) to form columns capable of spanning the hydrophobic region of the membrane (Figure 4D). Many columns would come together to form a barrel-stave channel. The cylindrical structure would be of variable size, depending on the number of columns. The observation that channels formed in planar phosphoglycerolipid membranes showed a remarkable preference for large conductance decrements that were multiples of 4 nS100 provided solid support for a cylindrical structure composed of columns. However, it also required that conductance decrements preferentially lost an even number of ceramide columns. This could be explained by the strong dipole formed when the 6 amide linkages are all oriented in the same direction in the column. Thus, adjacent columns would need to be oriented in opposite directions to minimize the energy of the dipole−dipole interactions (Figure 4D). Both negative stain electron microscopy and electrophysiological studies indicated that the most common ceramide channels formed 10 nm aqueous pores104 (∼50 columns), although preference for this size is weak as both larger and smaller channels were present in large numbers. Molecular dynamic simulations indicate that the proposed structure is stable.109 A 14-column channel, 6 ceramides per column, was placed into a palmitoyloleylphosphotidyl choline bilayer with an appropriate amount of water and ions. In the full-atom simulation, in 1 ns the interface between the channel and the surrounding bilayer adjusted itself so that a continuous polar surface facing the water phase was generated. This involved some distortion of the phospholipids at the interface with the channel so as to cover the hydrophobic tails of the ceramide molecules at the end of the columns. The channel, for its part, took on a somewhat hourglass shape in order to meet with the surrounding bilayer. The structure was stable until the end of the 10 ns simulation. The surface of the inner lining of the channel is composed of the twin hydroxyls from the polar end of ceramide and bridging water molecules. This hydroxyhydrogen-bonded network merges with the network of water molecules in the bulk phase.

The proper dispersal of ceramide into a mitochondrial suspension results in the permeabilization of the outer membrane to proteins.89,96 The elevation of mitochondrial ceramide levels does not cause an active secretion of proteins from the intermembrane space but rather a bidirectional flux as expected from a channel. Generally protein release is measured because of the vast difference in the volume of the intermembrane space and the extramitochondrial space. Often specifically cytochrome c release is measured because of its importance in apoptosome formation. However, the fact that other proteins (up to 100 kDa) are released almost simultaneously97,98 means that the release pathway must be large enough to release also much larger proteins. Regardless, when using isolated mitochondria one does not know what is forming the channel. However, ceramide also forms channels in phospholipid membranes that are long-lived and large enough to allow the translocation of even large proteins.99−103 The pore size of the typical channel was reported to be 10 nm in diameter but was also observed to be much larger.104 A pore diameter of 10 nm would have a protein molecular mass cutoff of 400 kDa. This estimate was obtained both from electrophysiological measurements and from negative stain electron microscopy of ceramide channels formed in phospholipid membranes.104 Thus, it is reasonable to conclude that ceramide would form similar channels in the mitochondrial outer membrane. Experiments on isolated mitochondria reported that the proteins released by ceramide had SDS peptides up to 60 kDa,89 but those investigators did not determine the cutoff as a function of ceramide dose. Still, the largest protein whose release was monitored during apoptosis, Smac-yellow fluorescent protein, has a monomer molecular mass of 50 kDa.98 Thus, ceramide channels are large enough to allow the release of relevant proteins from mitochondria For the ceramide channel hypothesis to be physiologically relevant, the self-assembly of hundreds of ceramide molecules to form a channel would have to occur at mole fractions of ceramide to phospholipid that are found in mitochondria within living cells at the time of outer-membrane permeabilization. The ceramide-induced permeabilization of the outer membrane in isolated mammalian mitochondria occurs at mole fractions of ceramide to phospholipid (starts at 0.2 mol %)95 that have been measured in the outer membrane of mitochondria from cells early in apoptosis. TNFα treatment raised ceramide levels to values ranging from 0.45 to 0.65 mol % depending on the cell type used.86,105 γ-Radiation yielded 0.4 mol percent ceramide.106 In these cells, mitochondrial ceramide levels become elevated early in the apoptotic process when the commitment step, protein release, takes place. Thus, without any other factors influencing the stability of the ceramide channels, channel formation should be taking place at the measured ceramide levels. However, a number of factors do influence the stability of ceramide channels,96,103,107,108 and the influence of these factors in combination has not been explored; thus, it is difficult to assess the propensity for channel formation under the conditions experienced in the outer mitochondrial membrane of living cells. Nevertheless, the fact that ceramide channel formation takes place at physiologically relevant ceramide levels argues for a role of these channels in the permeabilization of the outer membrane to proteins.

4.3. Gating of Ceramide Channels

The assembly of lipids into organized superstructures, save for the formation of crystals or separate phases, is a rather new process, and thus there is little existing information that can be used for guidance. Lipid channels, as a special type of superstructure, have the function of translocating matter across the membrane and changes in the ability to translocate matter could be regarded as gating. Unlike the gating described for many channels formed by proteins, lipid channels might be expected to gate by an assembly/disassembly process. Channelforming antibiotics such as monasomycin,110 nystatin,111 and gramicidin112 also seem to gate by an assembly/disassembly mechanism. For ceramide channels, such a mechanism makes sense, and a variety of substances seem to influence the apparent stability of the channels. In addition, changes in the steady-state level of ceramide will also “gate” the channel simply by mass action because the channel is in dynamic equilibrium with ceramide monomers or nonconducting ceramide H

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aggregates.100 The steady-state level is influenced by changes in enzymatic activity that increases ceramide production (ceramide synthase, dihydroceramide desaturase, sphingomyelinase, reverse ceramidase) or increase ceramide consumption (ceramidase, sphingomyelin synthase, glucosyl ceramide synthase).113 It is also influenced by ceramide compartmentation and the rate of translocation from one membrane to another.114 The stability of ceramide channels is influenced by the presence of other sphingolipids. The immediate precursor, dihydroceramide, strongly inhibits ceramide channel formation.103 Another precursor, sphingosine, also inhibits ceramide channel formation.107,115 The inhibition was seen also in ceramide channels formed in phospholipid membranes, demonstrating that the inhibitory effect is directly on the channels. In experiments on isolated mitochondria, addition of both sphingosine and ceramide resulted in low permeability, but this was enhanced by removal of sphingosine by the addition of fatty-acid-free albumin.107 The permeabilization resulted from the ceramide that was already present in the outer membrane. Mechanistically, these inhibitory effects can be understood in the context of the evidence that ceramide channels are highly organized, highly hydrogen-bonded structures.100 The incorporation of similar molecules into the structure could introduce destabilizing defects. The incorporation of sphingosine would also introduce positive charges that could destabilize by means of electrostatic interactions. Although cells could use these lipids to regulate ceramide channels, there is no indication that such a regulatory mechanism exists. However, the conversion of dihydroceramide to ceramide by the desaturase would both remove inhibition and increase ceramide levels; therefore, the process should contribute to a supralinear dependence of ceramide channel activity on ceramide mole fraction.

asymmetric carbons.117 However, this interaction is sensitive to the length of the acyl chain on ceramide. If the acyl chain is only 2 carbons long, this ceramide forms channels with the same potency as the typical 16-carbon chain ceramide found in cells. However, Bcl-xL has no effect on this short-chain ceramide’s ability to self-assemble into channels. Optimal sensitivity was reported with 16-, 18-, and 20-carbon chains. Far lower sensitivity was observed on channel formation by the 24carbon acyl chain ceramide (also an important physiological form of ceramide). These findings speak to the molecular mechanism of interaction but they also have physiological correlates. In human cells there are 6 ceramide synthases that have a marked preference for producing ceramides of specific chain lengths.118 Differential activation of specific ceramide synthases results in preferential production of ceramides of particular chain lengths. The 16- and 18-carbon chain ceramides are elevated in mitochondria early in the apoptotic process92,119 when the decision whether to release proteins from mitochondria is being made. Thus, at this time, the level of Bcl-xL is critical to this decision and the inhibitory effect is important. Later on, the 24-carbon version becomes elevated119 and presumably has other functions. Clearly, the actions of the apoptotic proteins have the characteristics of an important regulatory process. Evidence for the site on Bcl-xL to which ceramide binds points to the hydrophobic groove, critical to the ability of BclxL to bind to and neutralize pro-apoptotic proteins.117 This groove binds to a critical BH3 domain on Bax and Bak, preventing these from forming channels in the outer membrane.120,121 Drugs that also bind to this groove122−124 (2-methoxyantimycin A3, ABT-737, and ABT-263) interfere with the action of Bcl-xL: both its ability to bind to Bax and Bak and its ability to disassemble ceramide channels.117 Although indirect, the evidence indicates that the acyl chain of ceramide may be binding to the hydrophobic groove of Bcl-xL. Docking experiments support this conclusion. In sharp contrast to the action of antiapoptotic proteins, Bax acts synergistically with ceramide to permeabilize the outer membrane of isolated mitochondria.108 When doses of ceramide or activated Bax (either with octyl glucoside or with t-Bid) are used that produce low levels of permeabilization, the combination results in a more than additive permeabilizing effect. The enhancement by the addition of Bax was reduced and even eliminated when higher doses of ceramide were added to the mitochondria, resulting in higher levels of permeabilization. In addition, the dose of Bax that resulted in half-maximal enhancement was reduced as the permeabilization induced by ceramide was increased. This was interpreted as an increase in affinity of Bax for larger ceramide channels, probably optimal with a larger radius of curvature. This could explain the enhancement if Bax binding favored a larger radius of curvature. The results were also consistent with multiple Bax molecules binding to a single ceramide channel. Thus, the action of this pro-apoptotic protein is commensurate with its physiological role of permeabilizing the mitochondrial outer membrane. The effects of Bax on channel formation by ceramide analogues had a qualitatively different sensitivity to the sites of modification than was observed with Bcl-xL.117 The ability of Bax to enhance the permeabilization induced by the analogue was very sensitive to changes in the polar regions and to the chirality of the molecule. Bax had no effect on L-ceramide, the mirror image of natural ceramide, nor did it enhance Nmethylated ceramide. In contrast to Bcl-xL, the action of Bax

4.4. Regulation of Ceramide Channels

The ability of Bcl-2 family proteins to specifically influence the structure and stability of ceramide channels116 points to the existence of a regulation system that is maintained by natural selection to optimize the survival of the organism. The effects reported cannot be explained by nonspecific interactions or physical changes in the membrane environment. They can only be explained by specific binding interactions that result in structural changes that are propagated throughout the channel structure resulting in changes in stability or organization. Evidence for the destabilizing action of antiapoptotic proteins includes Bcl-xL, Bcl-2, and CED-9, although most of the published work focused on Bcl-xL.96,117 Addition of ceramide to mitochondria containing any of these proteins results in a reduction of the degree of permeabilization of the outer membrane to cytochrome c.96 This reduction in permeability was found to be dependent on the dose of added antiapoptotic protein. When subjected to a Hill analysis, equating the reduction in conductance to binding, the results were consistent with the formation of a 1:1 complex between BclxL or CED-9 and the ceramide channel. The same inhibitory effect was observed using isolated mitochondria from yeast (S. cerevisiae), providing confidence that the interaction is direct. Experiments on a defined system, planar phospholipid membranes, confirmed that the interaction is direct. The use of ceramide analogues showed that the ability of Bcl-xL to disassemble ceramide channels was rather tolerant to changes in the polar headgroup and in the chirality of the two I

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were ∼20 nm in diameter. The pores were ringed by raised areas consistent with being surface domains of the Bax proteins (Figure 5). It was estimated that 22 monomers were forming

was not sensitive to the length of the acyl chain. Thus, the site of Bax binding, as well as the mode of action, differs markedly from those of Bcl-xL. The diametrically opposite effects on ceramide channels of two members of the same protein family that have diametrically opposite effects on the apoptotic process must be more than a simple coincidence. 4.5. Ceramide Channels and Disease

No link between alteration in the formation or structure of ceramide channels and disease has been made. The difficulty is that ceramide is involved in many cellular functions, such that it is daunting to assign cause and effect with any degree of confidence. In addition, there is no direct evidence that ceramide channels are formed in the mitochondrial outer membrane in living cells. The lack of pharmacological tools and the mechanistic entanglement between ceramide channels and Bcl-2 family proteins make it difficult to decide what structure is responsible for the permeabilization of the outer membrane to proteins. However, when ceramide levels are elevated, they do favor apoptosis, and when these are diminished, the propensity for apoptosis is diminished. If this outcome is due to changes in the propensity for ceramide-channel formation, then a number of disease states could be attributed to these channels. For example,125 mutations in Drosophila in arrestin and phospholipase C, proteins involved in the phototransduction cascade, can lead to retinal degeneration. This phenotype was suppressed by the targeted expression of ceramidase, which reduced ceramide levels. Similar results were obtained in a retinal degeneration mouse model126 where ceramide levels increase during photoreceptor death. Here the application of myriocin, an inhibitor of ceramide synthase, inhibited the apoptotic cell death of the photoreceptors. Other forms of cell death such as hypoxia-induced neuronal apoptosis can be inhibited by reducing de novo ceramide synthesis. 127 Conversely, the lifespan of yeast is increased by the deletion of a ceramide synthase, longevity assurance gene 1.128 Finally, targeted delivery of ceramide was demonstrated to effectively kill an aggressive form of leukemia.129

Figure 5. Section analysis through a Bax channel formed in a phospholipid bilayer adsorbed to a mica surface. The membrane surface was scanned by atomic force microscopy, and a section through a channel is shown. The pore is surrounded by a raised ring (upper arrow) interpreted as protein domains raised above the membrane surface. The distance between the arrows includes the membrane thickness (4.5 nm) and the raised protein ring (2.4 nm). Reprinted with permission from ref 138. Copyright 2002 Elsevier B.V.

one channel. Channel formation depended on the addition of millimolar Ca2+. The formation of large pores (25−100 nm) in liposomes by t-Bid/Bax was demonstrated by two independent groups81,139 (Figure 6).

5. CHANNEL FORMATION BY BAX AND BAK The pro-apototic proteins, Bak and Bax, are generally believed to be responsible for the permeabilization of the mitochondrial outer membrane leading to the release of proteins.130−133 This is supported by a great deal of published work following the original studies of Wei et al.134 Apoptosis can still proceed with one of these proteins knocked out but is greatly inhibited when both are knocked out. However, as already pointed out, mitochondrial ceramide levels are also not elevated in the double knockout following an apoptotic signal because Bak is needed to activate the necessary ceramide synthase.92 In addition to channel formation, the Bcl-2 family proteins are also involved in mitochondrial fission and fusion, with fission being associated with apoptosis and fusion being associated with resistance to apoptosis.135 Interestingly, ceramide favors negative curvature and membrane vesiculation by budding.136,137 How these aspects of mitochondrial dynamics relate to outer-membrane permeabilization is not clear.

Figure 6. Electron micrographs of rapidly frozen liposomes without (control) and with Bax present (+Bax). In the presence of Bax, large holes were formed in the liposomes. Reprinted with permission from ref 139. Copyright 2010 American Society for Biochemistry and Molecular Biology.

5.2. Bax Channel Formation in Phospholipid Membranes

Electrophysiological recordings of Bax channels were first reported in 1997.140,141 Antonsson’s group,140 using Bax lacking 20 amino acids at the C-terminus, reported a variety of conductance levels. In a buffered solution (pH = 7) containing125 mM NaCl, they first observed a flickering 5.6 pS conductance that was overshadowed by a larger conductance fluctuating between 26 and 250 pS. They report further discrete conductance increments of multiples of 450 pS. This conductance favored Na+ over Cl− by 2:1. Schlesinger and co-workers141 also studied Bax lacking the C-terminal hydrophobic region. They report 300 pS conductances in 150 mM

5.1. Visualization of Bax Channels

Atomic force microscopy was used138 to visualize the channels formed by Bax on a phospholipid membrane spread out on a mica surface. Using membranes containing 50% cardiolipin, 100−300 nm diameter pores were observed. Using a 1:1 mixture of phosphotidyl choline/phosphotidyl serine, the pores J

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KCl but a preference for anions (Cl− over K+ by 2 or 3 to 1). The channels reported by both groups are most likely too small for protein translocation (expect for the very smallest protein, cytochrome c) unless the conducting events are actually substates of a much larger structure. The first group reported that voltage favored channel formation in the membrane, and the latter reported some degree of rectification. Neither reported any voltage gating. Electrophysiological studies of full-length Bax activated by treatment with octylglucoside revealed the formation of two types of conductances using the same Bax preparation and the same experimental technique.142 The outcome varied from one experiment to another for no apparent reason. Type A channels are well-behaved with a conductance of 4.5 nS in 1 M KCl and subconductance levels. Many of these channels can form in one membrane during an experiment (Figure 7A). These are

voltage-independent, and the transformation into type A channels results in gaining voltage-dependence. Type A channels are not affected by LaCl3 addition and seem to be unable to convert to type B. This directionality in conversion suggests that type B channels are inherently unstable. Perhaps the type B channels are those capable of releasing proteins from mitochondria and conversion to type A renders them incapable of protein release. This could represent one more level of control over the initiation of the execution phase of apoptosis. Bax channel formation in liposomes has been used to estimate the physical size of the Bax channel. Saito and coworkers146 analyzed the size based on the ability of dextrans to interfere with the efflux of FITC-labeled cytochrome c from liposomes induced by the addition of Bax lacking the Cterminal segment. They observed the greatest interference with a dextran 3 nm in diameter. An analysis of the dependence of efflux on the added amount of Bax yielded a curved Hill plot with a maximum slope of 4. They concluded that Bax formed a tetramer just large enough to allow the permeation of cytochrome c. Kuwana and co-workers147 found that, in the presence of cardiolopin, BH3-domain activated Bax can release 2 000 kDa dextran from liposomes, and this release is inhibited by Bcl-xL. They and others showed that t-Bid/Bax form large pores (25−100 nm) in liposomes containing cardiolipin. The use of GFP-Bax to estimate the size of the Bax/Bak complexes formed in the outer membrane of mitochondria in living cells148 yielded the conclusion that the fluorescent foci were composed of hundreds of Bax monomers. However, there is no evidence that the large fluorescent foci are the channels or that the entire structure is a channel. The authors conclude that the size of these foci precludes the existence of channels and thus must represent “leaks” in the membrane. This begs the question as to what is a leak and what is a channel. The use of Bax-derived peptides to study channel formation that might reflect the channel formed by Bax (see, for example, ref 149), is very risky because amphipathic peptides are known to easily form lipid-dominated toroidal pores. By disrupting the membrane structure and forming these lipidic pores, the use of these peptides determines rather than tests what might be the permeation pathway. Without the rest of the protein constraining the possible structures, how does one know whether the observations made have any meaning beyond being a biophysical curiosity?

Figure 7. Electrophysiological tracings of Bax channels formed in a planar phospholipid membrane. (A) Conductance increments following the addition of activated Bax to the aqueous phase bathing the planar membrane. (B) Current decrements following the application of a 70 mV potential to a membrane containing Bax channels. The inset shows a histogram of the sizes of the conductance decrements. The aqueous solution contained 1.0 M KCl buffered to pH 6.9. An extensive study of these channels was published in ref 142.

voltage-gated channels that close at 70 mV in 1.5 nS steps (Figure 7B). Closure is asymmetric in that it occurs only at one sign of the membrane potential, usually positive on the side of Bax addition. Thus, typically all the channels that form seem to be oriented in the same direction, and that direction varies from one experiment to another. This surprising orientation requires some form of strong cooperativity between channels. It was first reported for VDAC and called autodirected insertion.143,144 When the addition of Bax results in a large noisy conductance, this outcome is referred to as a type B channel.142 The conductance behaves as one single large channel. Addition of a few micromolar LaCl3 results in a drop in conductance to a new stable level after a stochastic delay consistent with one structure having to overcome an energy barrier. The remaining conductance is a population of channels indistinguishable from the type A channels. It is proposed that La3+ ions bind to the membrane lipids, increasing the lateral pressure of the hydrocarbon region,145 and this acts on the Bax channels, forcing the conversion of the one large channel into a population of small channels via a 2-dimensional budding process. This would be a 2-dimensional version of vesicles budding off a large vesicular structure. Type B channels are

5.3. Mitochondrial Apoptosis-Induced Channel

Induction of apoptosis by the removal of growth factor from cultured cells results in the formation of a high conductance channel in the mitochondrial outer membrane as detected by patch-clamping.150 This was named MAC, mitochondrial apoptosis-induced channel. Other treatments that induce apoptosis, such as staurosporine, also induce the formation of MAC in mitochondria.151 Overexpression of antiapoptotic proteins inhibit apoptosis and also inhibits MAC formation.150 MAC has a variable conductance generally ranging between 2.5 and 5 nS in 150 mM KCl. Dextran-exclusion experiments152 yielded a molecular mass cutoff between 17 and 45 kDa and an estimated pore diameter between 2.9 and 7.6 nm.153 This is somewhat smaller than the channels visualized by atomic force microscopy, but a larger structure might form under different conditions. MAC is reported to be essentially voltageindependent. Compelling evidence indicates that Bax and/or Bak are necessary components of MAC151,154 or are necessary for MAC to form. The presence of only one of these is K

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Table 1. Comparison of Conductances and Selectivites Reported for Bax and MAC Channels protein or named channel Bax (ΔTM)139 Bax (ΔTM)140 MAC149−153 Bax (full length) type A141 Bax (full length) type B141 a

conditions

conductance (nS)

conductance adjusted for 150 mM KCl (nS)a

ion selectivity Pcat+/PCl−

voltage dependence

0.03−0.5 0.3 1.5−5 0.8

2 0.32 3.1−3.7 3.6

no no no yes

10−100

N/Db

no

125 mM NaCl 0.026, 0.25, 0.45 150 mM KCl 0.3 150 mM KCl 1.5−5 1 M KCl 4.5 (plus 1.5 nS sub conductances) 1 M KCl 60−100 s

Calculated assuming linearity with ionic activity. bN/D = not determined.

to proteins by ceramide and Bax/Bak channels is only obvious during apoptosis. Yet, one might expect that the components of these channels that signify the readiness of the cell to live or die are likely to fluctuate in quantity in the outer membrane under normal and stressed conditions, only truly manifesting themselves when a decision to undergo apoptosis is made. How often, during its life, does a cell go to the brink before returning to a normal state? Both genetic factors and the physical/chemical state of the cell and its environment predispose cells to be sensitive or resistant to apoptotic stimuli and thus affect the likelihood of entering pathological conditions. The surprising results of knocking down VDAC genes indicate both the multiple functions of VDAC proteins and how disregulation of the flux of matter through the outer membrane has pronounced consequences all the way to the level of animal behavior. Perhaps the major overall lesson to be gained form this research is not to allow the paradigms that quickly coalesce to limit one’s perspective. The study of mitochondrial channels will likely continue to yield more surprises.

sufficient to observe the channel. MAC-like activity was detected in yeast cells expressing Bax,150 demonstrating that MAC formation does not require other Bcl-2 family proteins. Because MAC is detected either by patch-clamping mitochondrial membranes or transferring material from such membranes to liposomes, its composition has not been completely defined. A comparison of some of the electrophysiological properties reported for Bax and MAC channels (Table 1) shows both agreement and differences. The differences may arise from the way Bax is isolated and the experimental conditions used. However, the complexity of the Bax channel itself may be partly to blame. 5.4. An Integrated View of Bax/Bak Channels?

As with most investigations, each experimental approach used to study Bax/Bak channels has yielded insights into these channels from a limited perspective. The study of MAC channels yields information that is closest to the channel found in vivo. The use of biochemical155 and structural methods have provided information on channel size and how these proteins change structure from the simple soluble protein to the conformation that forms the structure of the channel. The reconstitution of Bax channels from defined components has yielded information on what the pure Bax protein can do on its own given the appropriate membrane environment. The ability of ceramide to interact with and act synergistically with Bax to permeabilize the outer membrane to proteins demonstrates the existence of an added dimension of complexity. All these studies have overlapping threads, but these are still too tenuous to be able to construct an integrated picture of these channels and the various forms that they can take. The high level of complexity already identified indicates that these channels are influenced and regulated at many levels, mirroring the complex system that regulates the initiation and progression of apoptosis.

AUTHOR INFORMATION Corresponding Author

*Phone: 301-405-6925. Fax: 301-314-9358. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biography

6. CONCLUSION AND PERSPECTIVES It took a long time from its discovery156 for the importance of the mitochondrial outer membrane to be appreciated. Rather than being merely vestigial, the outer membrane is now recognized as being essential if only to prevent the cell from spontaneously undergoing apoptosis. More likely the appreciation for the outer membrane’s influence on cellular functions in health and disease will continue to grow as more regulatory pathways come to light. The channels in this membrane are the main conduits for the flow of matter, and the high degree of regulation of these channels clearly demonstrates that the role of these channels is not merely to provide flux of matter but to control this flux in harmony with changes in cellular physiology. The regulation of metabolite flux by VDAC channels is probably taking place constantly whereas the permeabilization

Marco Colombini was born in Modena, Italy, but grew up in Montreal, Canada. He received his Ph.D. in Biochemistry (1974, McGill University) under Rose Johnstone and was the first to study Na+gradient powered active transport of amino acids in plasma membrane vesicles. During his postdoctoral studies (1974−1976) with Alan Finkelstein at Albert Einstein College of Medicine (AECOM, Bronx, L

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NY), he was codiscoverer of VDAC channels in mitochondria, the first intrinsic channels to be studied at the single-channel level. After serving as assistant professor at AECOM until 1979, he joined the faculty of the University of Maryland (College Park) first in the Department of Zoology and then Biology, and this is his current professorial appointment. He has affiliate appointments with the Department of Cell Biology and Molecular Genetics (2001−current) and the Department of Chemistry and Biochemistry (2004−current). He served as Associate Chair and Director of Graduate Studies for Biology (2006−2012), as Chair of the Bioenergetics Subgroup of the Biophysical Society (2004−2007), and on the governing Council of the Biophysical Society (2008−2012). He is on the editorial board of Biochimica et Biophysica Acta, Biomembranes, and the Journal of Bioenergetics and Biomembranes. In addition to his long-standing pioneering research on VDAC, his lab discovered ceramide channels, the first stable channels formed solely by lipids. Current research centers on the roles of channels in the mitochondrial outer membrane in initiating the execution phase of apoptosis. Currently his H-index is 47.

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