Planar Phospholipid Membranes Research Articles

Cooperativity and Steep Voltage Dependence in a Bacterial Channel.

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.

Formation and dynamics of endoplasmic reticulum-like lipid nanotube networks.

We report on the self-organized formation and dynamics of artificial lipid nanotube networks, which, in terms of morphology and behavior, resemble the endoplasmic reticulum(ER) of biological cells. The networks, initially generated from a solid-supported planar phospholipid membrane, undergo a morphological transformation, triggered by the chelation and removal of Ca2+ from the environment surrounding the membrane. Calcium depletion gradually causes de-pinning, thus de-wetting, at the membrane-substrate interface. We observe dynamic re-arrangements very similar to the ones reported for the cellular ER, such as sliding of tubes and formation of new junctions, and quantify these transformations. We also show occurrences of the dynamic replacement of lipidic particles on nanotubes as indicators for the existence of a tension gradient throughout the network, as well as the spontaneous formation of small vesicles from semi-free floating tubes. We propose that these artificial networks are suitable to serve as a bottom-up-generated structural model for the cellular ER, whose fascinating characteristic morphology is suggested to be tied to its biological function, but with respect to formation, dynamics, and functional details still incompletely understood.

Formation of planar unilamellar phospholipid membranes on oxidized gold substrate.

Supported planar phospholipid membranes are used in a range of biophysical measurements, typically for characterizing protein-membrane interactions. Liposome deposition is the most common method to create such membranes. The ability of liposomes to fuse into a lamellar membrane during deposition is strongly dependent on the surface chemistry; some important substrate materials such as oxidized gold do not promote liposome fusion. Circumventing this determinism poses an enduring challenge to membrane biophysics. Here, the authors show that the effect of surface chemistry can be overcome by using osmotic stress. Reproducible single bilayer coverage was achieved on oxidized gold surface from liposomes of a variety of lipid compositions, as demonstrated by quartz crystal microbalance measurements and confirmed via fluorescence microscopy imaging. The continuity of the deposit was confirmed by fluorescence recovery after photobleaching. Using mixtures of di-myristoyl and di-palmitoyl lipids, it was also demonstrated that the formation of fused lamellar membranes upon osmotic stress is a sensitive function of the thermodynamic phase of the membrane.

Ceramide channels: destabilization by Bcl-xL and role in apoptosis

Ceramide is a bioactive sphingolipid involved in mitochondrial-mediated apoptosis. Our data suggest that ceramides directly regulate a key initiation step in apoptosis: mitochondrial outer membrane permeabilization (MOMP). MOMP allows release of intermembrane space proteins to the cytosol, inducing the execution of the cell. Ceramides form channels in planar phospholipid membranes and outer membranes of isolated mitochondria, channels large enough to facilitate passage of proteins released during MOMP. Bcl-xL inhibits MOMP in vivo and inhibits the formation of ceramide channels in vitro. However the significance of Bcl-xL's regulation of ceramide channel formation within cells was untested. We engineered Bcl-xL point mutations that specifically affect the interaction between ceramide and Bcl-xL to probe the mechanism of ceramide channel regulation and the role of ceramide channels in apoptosis. Using these mutants and fluorescently-labeled ceramide, we identified the hydrophobic groove on Bcl-xL as the critical ceramide binding site and regulator of ceramide channel formation. Bcl-xL mutants with weakened interaction with ceramide also have reduced ability to interfere with ceramide channel formation. Some mutants have similar altered ability to inhibit both ceramide and Bax channel formation, whereas others act differentially, suggesting distinct but overlapping binding sites. To probe the relative importance of these channels in apoptosis, Bcl-xL mutant proteins were stably expressed in Bcl-xL deficient cells. Weakening the inhibition of either Bax or ceramide channels decreased the ability of Bcl-xL to protect cells from apoptosis in a stimulus-dependent manner. These studies provide the first in vivo evidence for the role of ceramide channels in MOMP.

A Highly Cooperative and Steeply Voltage Gated Channel Triplet

When reconstituted into planar phospholipid membranes, a 4.5nS (in 1M KCl) membrane channel complex behaves as if it is composed of three channels that are operating in a highly cooperative manner. The channels are steeply voltage-gated (e-fold for 1.8 mV) and well-organized. In the ± 70mV range, no voltage gating takes place until the first channel, channel 1, closes at high positive potentials (>70 mV). Only following this closure does channel 2 begin to gate in response changes in voltage. For channel 2, closure takes place at low negative potentials (typically −25 to −30 mV). It is only when channel 2 is closed that channel 3 closes routinely but does so at low positive potentials (typically 25-30 mV). Simultaneous reopening of channels 2 and 3 is common, demonstrating especially high cooperativity. These and other remarkable properties support a working model for the gating and cooperativity of these channels. The highlights include anti-parallel orientation of the channels and the voltage sensor dipole being responsible both for voltage gating and cooperativity. (Supported by NSF grant MCB-1023008)

Bax channel triplet: co-operativity and voltage gating

Bax, despite being a cytosolic protein, has the distinct ability to form channels in the mitochondrial outer membrane, which are capable of releasing proteins that initiate the execution phase of apoptosis. When studied in a planar phospholipid membrane system, full-length activated Bax can form conducting entities consistent with linearly organized three-channel units displaying steep voltage-gating (n=14) that rivals that of channels in excitable membranes. In addition, the channels display strong positive co-operativity possibly arising from the charge distribution of the voltage sensors. On the basis of functional behaviour, one of the channels in this functional triplet is oriented in the opposite direction to the others often resulting in conflicts between the effects of the electric field and the positive co-operativity of adjacent channels. The closure of the first channel occurs at positive potentials and this permits the second to close, but at negative potentials. The closure of the second channel in turn permits closure of the third, but at positive potentials. Positive co-operativity manifests itself in a number of ways including the second and the third channels opening virtually simultaneously. This extraordinary behaviour must have important, although as yet undefined, physiological roles.

Cobaltabisdicarbollide Macroanion is able to Diffuse across the Lipid Membrane; Study of Kinetics and Transport

The ability to form membranes is not restricted to polar lipid molecules or even to organic molecules, as the boron-based molecule cobaltabisdicarbollide, [Co(C2B9H11)2]-, (commonly known as cosan) can also form vesicles and membranes. Here we show that cosan and its derivatives can interact with phospholipid membranes.Cosan is a monoanionic metallabisdicarbollide, which is soluble in both water and oils. Structurally, cosan has no similarity to the polarized lipid molecules that make up biological membranes. It comprises a cobalt atom sandwiched by two carboranyl clusters [1-3]. Although these clusters are hydrophobic, the metal ion imparts a dispersed negative charge spread over the whole molecule (fig.1). As a consequence of this, the exposed B-H and C-H bonds of the carboranyl clusters possess weak dipoles and form intermolecular attractions that give the molecule its unusual physico-chemical property of being simultaneously hydrophobic and hydrophilic. This duality allows cosan to interact with lipid membranes.Using membrane electrophysiology recordings and direct measurements of COSAN concentrations by inductively coupled plasma mass spectrometry (ICP-MS) we show that COSAN transits cell-free artificial lipid membranes. We study the kinetics of the transport and try to get insights on the mechanism by which COSAN crosses planar phospholipid membranes.[1] C.E. Housecroft et al., Encyclopedia of Inorganic Chemistry. John Wiley & Sons; 2008.[2] M.F. Hawthorne et al, J. Am. Chem. Soc 1968;90:879-896.[3] R.N. Grimes, Carboranes. Academic Press: Burlington, MA; 2011.

S.27.01 Mitochondria and Aβ metabolism

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm, and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. For example, one of the long-standing puzzles was that in permeabilized cells, adenine nucleotide translocase is less accessible to cytosolic ADP than in isolated mitochondria. Still another puzzle was that, according to channel-reconstitution experiments, voltage regulation of VDAC is limited to potentials exceeding 30 mV, which are believed to be much too high for MOM. We have solved these puzzles and uncovered multiple new functional links by identifying a missing player in the regulation of VDAC and, hence, MOM permeability — the cytoskeletal protein tubulin. We have shown that, depending on VDAC phosphorylation state and applied voltage, nanomolar to micromolar concentrations of dimeric tubulin induce functionally important reversible blockage of VDAC reconstituted into planar phospholipid membranes. The voltage sensitivity of the blockage equilibrium is truly remarkable. It is described by an effective “gating charge” of more than ten elementary charges, thus making the blockage reaction as responsive to the applied voltage as the most voltage-sensitive channels of electrophysiology are. Analysis of the tubulin-blocked state demonstrated that although this state is still able to conduct small ions, it is impermeable to ATP and other multi-charged anions because of the reduced aperture and inversed selectivity. The findings, obtained in a channel reconstitution assay, were supported by experiments with isolated mitochondria and human hepatoma cells. Taken together, these results suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC interaction with tubulin at the mitochondria–cytosol interface. Immediate physiological implications include new insights into serine/threonine kinase signaling pathways, Ca2 + homeostasis, and cytoskeleton/microtubule activity in health and disease, especially in the case of the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. In the present review, we speculate how these findings may help to identify new mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs, and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect). This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Type a Bax Channels: The Highly Voltage-Gated Form of this Killer Protein

Bax, a pro-apoptotic protein, translocates from the cytosol to the mitochondrial outer membrane (MOM) where it oligomerizes and forms channels. When reconstituted into planar phospholipid membranes, Bax forms two types of channels: Type A and Type B. The former is voltage-gated and is the focus of this presentation. Electrophysiological studies of a single Type A channel show a complex gating pattern: a trimer of conductance decrements each with distinct voltage gating. The results are interpreted in terms of a linear trimer of strongly interacting subunit channels with the middle subunit oriented in the opposite direction from the others. The closing of the first subunit occurred around +70 mV with slow kinetics and steep voltage dependence. The second subunit didn't gate until the first one closed, after which, it closed with n = 13 and V0 ∼ −22 mV. Only with the second subunit closed, did the third subunit can start to gate. It closed with n = 32 and V0 ∼ +25 mV. Note that all the gating events are extremely voltage-dependent probably due to the oligomeric nature of the subunit channel. The restricted gating indicates that the gating domains are restricted by the state of neighboring subunits. Whereas the first and third subunits closed at positive voltage, the second subunit closed at negative indicating an opposite orientation. Adjacent trimeric channels provide opportunities for further interaction. A higher n value (20) was observed for gating of the second subunit in multi-channel membranes. This complex functional behavior provides insights into the properties and interactions of Bax proteins in membranes. These properties likely contribute to the decision-making process leading either to the formation large Bax channels and apoptosis or small Bax channels and cell survival. (supported by NSF grant MCB-1023008)

Role of charge screening and delocalization for lipophilic cation permeability of model and mitochondrial membranes

The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C12TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C10TEA and C16TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C12TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C12TPP induced H+ permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663–668). C12TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6μM. Besides, C12TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C10TEA and C16TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C12TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles.

Type a Bax Channels: Electrophysiological Properties

The pro-apoptotic protein, Bax, begins life as a soluble monomeric protein but can be activated to oligomerize and form two types of membrane channels: Type A channels are small and voltage-gated whereas Type B form larger channels and are voltage independent. To understand the properties that arise solely from the Bax protein and its interaction with a phospholipid membrane, Bax channels were formed and studied in planar phospholipid membranes. The Type A channels formed are oriented so that voltage-dependent closure occurs only at one sign of the membrane potential. An entire channel population responds in like manner but the sign of the voltage that results in voltage-gating differs from one experiment to another indicating that channel formation/insertion follows a form of auto-directed insertion as was first described for VDAC channels. Voltage-gating typically occurs at 70 mV resulting in a uniform population of 1.4 nS decrements. Three of these decrements appear to be associated with a single channel. Channel reopening/reassembly is a slow, multi-exponential process with the rate dependent on how long the channels were held closed. The use of triangular voltage waves reveals a complex behavior incompatible with the standard analysis applied to voltage-gated channels. These channels are unlikely to be responsible for protein release from mitochondria. Type B channels are likely to contribute to that process. The Type A channels may represent an alternative structure formed by activated Bax when cellular conditions do not quite warrant progression to the execution phase of apoptosis. (Supported by NSF grant: MCB-1023008).

Novel Mitochondria-Targeted Antioxidants: Plastoquinone Conjugated with Cationic Plant Alkaloids Berberine and Palmatine

To develop effective mitochondria-targeted antioxidants composed entirely of natural constituents. Novel mitochondria-targeted antioxidants were synthesized containing plant electron carrier and antioxidant plastoquinone conjugated by nonyloxycarbonylmethyl residue with berberine or palmatine, penetrating cations of plant origin. These compounds, SkQBerb and SkQPalm, were tested in model planar phospholipid membranes and micelles, liposomes, isolated mitochondria and living cells. SkQBerb and SkQPalm penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in mitochondria isolated or in living human cells in culture. Reduced forms of SkQBerb and SkQPalm as well as C10Berb and C10Palm (SkQBerb and SkQPalm analogs lacking plastoquinol moiety) revealed radical scavenging activity in lipid micelles and liposomes, while oxidized forms were inactive. In isolated mitochondria and in living cells, berberine and palmatine moieties were not reduced, so antioxidant activity of C10Berb and C10Palm was not detected. SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations; their prooxidant effect was observed at 1,000 times higher concentrations. In human cell cuture, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. This is the first successful attempt to construct mitochondria-targeted antioxidants composed entirely of natural components, namely plastoquinone, nonyl, acetyl and berberine or palmatine residues.

Dynamic Study of Ceramide Channels with a Microfluidic System

A novel microfluidic system enabling effective and robust dynamic perfusion of reagents directly to an on-chip phospholipid bilayer membrane was developed to study membrane channels. Here we report its application to the study of the dynamics of ceramide channels. The microfluidic system consists of a planar phospholipid membrane (PPM) formed across a 60-100 µm diameter aperture in a polyvinylidene chloride film formed within a thermoplastic polycarbonate substrate containing a microfabricated fluidic network. The volume of the network before the aperture is 2.3µL and the chamber beneath the aperture is 20nL. Constant pumping rates up to 5µL/min can be used without disrupting the fragile membranes and so the solution bathing the membrane can be completely replaced in less than 1 minute. Transmembrane current is monitored continuously under voltage-clamp conditions. For ceramide channel experiments, the membrane-forming solution was 5 mg diphytanoyl PC, 5 mg asolectin, 0.5 mg cholesterol, 0.13 mg C16-ceramide in 1 ml of hexanol/hexadecane (v/v 10:1). The sphingolipid, ceramide, self-assembles into large channels in phospholipid membranes and is known to play an important role in apoptosis. In our studies, channel dynamics were observed by inducing disassembly and reassembly of ceramide channels formed spontaneously in the PPMs. Perfusion with La3+ resulted in rapid disassembly. Channel reassembly occurred after washing away the La3+ using EDTA. Multiple cycles of disassembly and reassembly could be performed on the same membrane. Similar studies were performed by perfusing in Bcl-xL, a potent apoptosis inhibitor. Bcl-xL disassembles the channels rapidly but the reassembly is slow. The results are consistent with ceramide channels existing in equilibrium with ceramide monomers or aggregates in the membrane and this equilibrium can be shifted by interaction with chemical agents. Supported by grants from NIH (R21EB009485) and NSF (MCB-0641208).

Bax Forms Two Types of Channels, One is Voltage-Gated

Bax is a proapoptotic protein that self-assembles to form channels on the mitochondrial outer membrane, permeabilizing it, releasing cytochrome c to trigger apoptosis. However, the detailed mechanism of how Bax forms these channels and their dynamics are still unknown. We formed Bax channels in planar phospholipid membranes by adding octylglucoside-activated full length Bax to one side of the membrane. We found that Bax can form two different kinds of channels with different characteristics. The first kind (Type A) shows discrete, uniform-sized, step-wise conductance changes characteristic of single channels. These are voltage-gated: they close at high positive voltages (on the side of Bax addition) and open at low voltages. The second kind of conductance (Type B) consists of a noisy increase in current that does not appear to be composed of a population of channels but to be one large assembly. This conductance is not voltage-gated. However, upon the addition of 5µM La3+, the conductance decreases quickly after a variable delay, leaving a residual conductance of Type A channels. The channels in the residual conductance have the same voltage-gating characteristics and conductance as Type A. We speculate that the large channel undergoes 2-dimensional budding forming a population of small channels. This “budding” phenomenon is hypothesized to be the result of an interaction between La3+ and the lipids because the selectivity of Bax does not change after addition of La3+. The selectivity of both of these conductances favors cations unlike that reported for a truncated form of Bax, ΔC19 Bax. Supported by a grant from NSF (MCB-0641208).

Ceramide and activated Bax act synergistically to permeabilize the mitochondrial outer membrane

A critical step in apoptosis is mitochondrial outer membrane permeabilization (MOMP), releasing proteins critical to downstream events. While the regulation of this process by Bcl-2 family proteins is known, the role of ceramide, which is known to be involved at the mitochondrial level, is not well-understood. Here, we demonstrate that Bax and ceramide induce MOMP synergistically. Experiments were performed on mitochondria isolated from both rat liver and yeast (lack mammalian apoptotic machinery) using both a protein release assay and real-time measurements of MOMP. The interaction between activated Bax and ceramide was also studied in a defined isolated system: planar phospholipid membranes. At concentrations where ceramide and activated Bax have little effects on their own, the combination induces substantial MOMP. Direct interaction between ceramide and activated Bax was demonstrated both by using yeast mitochondria and phospholipid membranes. The apparent affinity of activated Bax for ceramide increases with ceramide content indicating that activated Bax shows enhanced propensity to permeabilize in the presence of ceramide. An agent that inhibits ceramide-induced but not activated Bax induced permeabilization blocked the enhanced MOMP, suggesting that ceramide is the key permeabilizing entity, at least when ceramide is present. These and previous findings that anti-apoptotic proteins disassemble ceramide channels suggest that ceramide channels, regulated by Bcl-2-family proteins, may be responsible for the MOMP during apoptosis.

Penetrating cation/fatty acid anion pair as a mitochondria-targeted protonophore

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H(+)-conducting fatty acid cycling mediated by penetrating cations such as 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C(12)TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (DeltapH) to a membrane potential (Deltapsi) of the Nernstian value (about 60 mV Deltapsi at DeltapH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C(12)TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H(2)O(2) formation. In intact yeast cells, C(12)TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.

Bax Enhances the Permeabilization of the Mitochondrial Outer Membrane Induced by Ceramide Channels: Implications on the Regulation of the Initiation of Apoptosis

Background: Bax is a pivotal pro-apoptotic Bcl-2 family protein that localizes to the mitochondrial outer membrane (MOM) during apoptosis and causes MOM permeabilization to proteins (MOMP). Earlier studies have demonstrated that upon an apoptotic stimulus, ceramide levels often greatly increase in cell membranes, including in the MOM. Elevation of ceramide in the MOM is sufficient to cause MOMP without requiring Bcl-2 family proteins. Moreover ceramide induced MOMP is reversed/prevented by the anti-apoptotic protein, Bcl-xL. Methods: Using rat liver or yeast mitochondria, the MOMP was measured with a dynamic cytochrome c accessibility assay. Ceramide's channel-forming ability was also assessed using a defined system: planar phospholipid membranes. Only C16-ceramide was used. Results: We found that Bax induces MOMP by apparently enlarging ceramide channels. While ceramide forms channels independently of Bax, the permeabilization is enhanced by the addition of less than 5nM oligomeric Bax. As much as 50nM oligomeric Bax alone did not result in any significant MOMP. The Bax enhancement occurs with an apparent affinity that increases with an increase in ceramide-induced MOMP, indicating an underlying mechanism by which Bax enhances ceramide-induced MOMP. Bax also causes apparent ceramide channel enlargement in yeast mitochondria, which lack Bcl-2 family proteins, as well as in planar phospholipid membranes, which is a defined, protein free, system. By contrast, monomeric Bax has no effect on ceramide channels in the aforementioned systems. The Bax inhibitor, Bci2 [Bruno Antonsson], prevents Bax mediated channel enlargement but does not affect permeabilization induced by ceramide alone. Conclusions: Both pro- and anti-apoptotic proteins regulate ceramide channels, consistent with ceramide channels being the pathway by which proteins are released by mitochondria early in apoptosis. (Supported by NSF grant: MCB-0641208)

VDAC Regulation by Cytosolic Proteins

Voltage-dependent anion channel VDAC, positioned on the interface between mitochondria and the cytosol, is at the control point of mitochondria life and death. This large channel plays the role of a “switch” that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. It controls them by switching between the open and “closed” states that are virtually impermeable to ATP and ADP. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptosis when cytochrome c and other apoptogenic factors are released from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals without direct involvement of VDAC in the permeability transition pore or hypothetical Bax-containing cytochrome c permeable pores. Closure of VDAC induced by such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin is compared to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. Tubulin strikingly increases voltage sensitivity of VDAC reconstituted into planar phospholipid membrane and could induce VDAC closure at < 10 mV transmembrane potentials. Experiments with isolated mitochondria confirm a tubulin-induced VDAC closure. Our findings suggest a novel mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria-cytosol interface. Overall, we demonstrate that VDAC gating is not just an observation made under artificial conditions of channel reconstitution but is a major mechanism of MOM permeability control.

Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration

Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation, dependent on the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent K(m) for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria-cytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail (CTT) peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in CTT throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character are only found in cells that do not have mitochondria.

10.1007/s11827-008-1009-4

Ionophoric activities of an N-terminus truncated gramicidin A (gA) analogue (mini-gramicidin) and its covalent dimer were studied in planar bilayer phospholipid membranes (BLM) using macroscopic current measurements (at high concentrations of the peptides) and single-channel recordings. As with gA-induced currents, mini-gramicidin-stimulated macroscopic currents through BLM underwent sensitized photoinactivation, i.e. were suppressed after irradiation with visible light in the presence of a photosensitizer generating singlet oxygen. The sensitivity of the tested compounds to photoinactivation descended in the following order: minigramicidin dimer > mini-gramicidin monomer > gA. The data from single-channel measurements and kinetic analysis of flash-induced photoinactivation obtained at different levels of macroscopic currents suggest that mini-gramicidin and its covalent dimer induce a variety of conducting states; their ratio depends on membrane thickness. Analysis of natural (mitochondrial and erythrocyte) membranes established that ionophoric activities of mini-gramicidin and its covalent dimer depend essentially on the membrane type.