Lipid Pore Research Articles

Transmembrane molecular transport during versus after extremely large, nanosecond electric pulses

Recently there has been intense and growing interest in the non-thermal biological effects of nanosecond electric pulses, particularly apoptosis induction. These effects have been hypothesized to result from the widespread creation of small, lipidic pores in the plasma and organelle membranes of cells (supra-electroporation) and, more specifically, ionic and molecular transport through these pores. Here we show that transport occurs overwhelmingly after pulsing. First, we show that the electrical drift distance for typical charged solutes during nanosecond pulses (up to 100 ns), even those with very large magnitudes (up to 10 MV/m), ranges from only a fraction of the membrane thickness (5 nm) to several membrane thicknesses. This is much smaller than the diameter of a typical cell (∼16 μm), which implies that molecular drift transport during nanosecond pulses is necessarily minimal. This implication is not dependent on assumptions about pore density or the molecular flux through pores. Second, we show that molecular transport resulting from post-pulse diffusion through minimum-size pores is orders of magnitude larger than electrical drift-driven transport during nanosecond pulses. While field-assisted charge entry and the magnitude of flux favor transport during nanosecond pulses, these effects are too small to overcome the orders of magnitude more time available for post-pulse transport. Therefore, the basic conclusion that essentially all transmembrane molecular transport occurs post-pulse holds across the plausible range of relevant parameters. Our analysis shows that a primary direct consequence of nanosecond electric pulses is the creation (or maintenance) of large populations of small pores in cell membranes that govern post-pulse transmembrane transport of small ions and molecules.

A lipocentric view of peptide-induced pores

Although lipid membranes serve as effective sealing barriers for the passage of most polar solutes, nonmediated leakage is not completely improbable. A high activation energy normally keeps unassisted bilayer permeation at a very low frequency, but lipids are able to self-organize as pores even in peptide-free and protein-free membranes. The probability of leakage phenomena increases under conditions such as phase coexistence, external stress or perturbation associated to binding of nonlipidic molecules. Here, we argue that pore formation can be viewed as an intrinsic property of lipid bilayers, with strong similarities in the structure and mechanism between pores formed with participation of peptides, lipidic pores induced by different types of stress, and spontaneous transient bilayer defects driven by thermal fluctuations. Within such a lipocentric framework, amphipathic peptides are best described as pore-inducing rather than pore-forming elements. Active peptides bound to membranes can be understood as a source of internal surface tension which facilitates pore formation by diminishing the high activation energy barrier. This first or immediate action of the peptide has some resemblance to catalysis. However, the presence of membrane-active peptides has the additional effect of displacing the equilibrium towards the pore-open state, which is then maintained over long times, and reducing the size of initial individual pores. Thus, pore-inducing peptides, regardless of their sequence and oligomeric organization, can be assigned a double role of increasing the probability of pore formation in membranes to high levels as well as stabilizing these pores after they appear.

A proposed Means to Evaluate Liposomes Mimicking Physiological Complexity for Fusion Potential

Cell signaling pathways rely on lipid vesicles to ferry signaling molecules within the confines of the cell, and when the signal needs to be conveyed, this vesicle fuses with the cell membrane to release its contents. The merger of these two lipid bilayers, and the formation of a lipidic fusion pore, is mediated by calcium ion and a number of proteins specific to this task. In neurons, Synaptotagmin I is a calcium ion binding protein found tethered to neurotransmitter vesicles, it is not completely understood how Synaptotagmin I interacts with the vesicle to facilitate the fusion of these lipid bilayers.

A Continuum Variational Approach to Vesicle Membrane Modeling

Biological membranes remodel in lipid pore formation, fusion, endocytosis and other processes. Traditionally, continuum membrane mechanics has been used to describe the physics of these remodelings. Membrane mechanics is a conservative, equilibrium theory and so cannot, a priori, describe the time course, flows and dissipations of a real system. Over the past few decades, physical scientists and mathematicians have developed global multi-physics field equations that describe the time course of processes for condensed matter in a thermodynamically consistent way. We use these equations to describe the membrane during lipid bilayer membrane remodelings. We analyze the vesicle membrane and its lipid layers as a bulk continuum variable in a Hamiltonian. The Hamiltonian includes the surface tension and curvature effects of the classical Helfrich model. The representations are, however, more flexible and can readily account for multicomponent systems, inhomogeneities, and changes in topology. Coupling the Hamiltonian to the motion of the aqueous medium with Rayleigh dissipation leads to a complicated, self-consistent system of partial differential equations that is solved numerically. Numerical schemes, designed specifically for this field theory, provide the position, velocity and forces of the fluid--vesicle system at each point in space and time. Classical models assume a specific shape for the vesicle (e.g., a sphere). The assumed shape will occur in the real world, however, only if it is a self-consistent solution of the equations. Our calculations yield values of all key variables and energies over time-the shape is an output. Movies that precisely illustrate the time evolution of the membrane configuration are generated. Changes over time are appreciated visually without reference to the equations--or even to the physics--of the remodeling processes.

Palmitic Acid Induces the Opening of a Ca2+-Dependent Pore in the Plasma Membrane of Red Blood Cells: The Possible Role of the Pore in Erythrocyte Lysis

Earlier we found that in the presence of Ca(2+) palmitic acid (Pal) increases the nonspecific permeability of artificial (planar and liposomal) membranes and causes permeabilization of the inner mitochondrial membrane. An assumption was made that the mechanism of Pal/Ca(2+)-induced membrane permeabilization relates to the Ca(2+)-induced phase separation of Pal and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer. In this article, we continue studying this pore. We have found that Pal plus Ca(2+) permeabilize the plasma membrane of red blood cells in a dose-dependent manner. The same picture has been revealed for stearic acid (20 μM) but not for myristic and linoleic acids. The Pal-induced permeabilization of erythrocytic membranes can also occur in the presence of Ba(2+) and Mn(2+) (200 μM), but other bivalent cations (200 μM Mg(2+), Sr(2+), Ni(2+), Co(2+)) are relatively ineffective. The formation of Pal/Ca(2+)-induced pores in the erythrocytic membranes has been found to result in the destruction of cells.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

ChemInform Abstract: Transdermal Delivery of Drugs and Enhancement of Percutaneous Absorption

This paper describes 1) the drug delivery through the skin to produce systemic effects, 2) the enhancement of percutaneous absorption by absorption enhancers, heating and complex formation, 3) the mechanism for the enhancement effect by enhancers, 4) the percutaneous absorption of peptides, and 5) the pharmacokinetic analysis for percutaneous absorption. 1,3-Dinitroglycerin, indomethacin (IND) and many drugs were efficiently absorbed via rat and rabbit skins in the presence of some enhancers, and using a microporous membrane therapeutic plasma concentrations were maintained for a long time. Enhancement of percutaneous absorption by the complex formation with fatty acid was observed for propranolol (PL) in vitro and in vivo. Heating at 42-45 degrees C also enhanced the percutaneous absorption dramatically, with decreased activation energies. The following mechanisms for the enhancement effect by enhancers were found: a) an increase in the fluidity of the stratum corneum lipids and reduction in the diffusional resistance to permeants, b) the removal of intercellular lipids and dilation between adherent cornified cells, c) an increase in the thermodynamic activity of drugs in vehicles, d) the exfoliation of stratum corneum cell membranes, the dissociation of adherent cornified cells and elimination of the barrier function. Peptides such as enkephalin, elcatonin and insulin were effectively absorbed through the skin in the presence of some enhancers and specific inhibitors, with no proteolytic degradation. The pharmacokinetic model with two parallel absorption processes, lipidic and aqueous pore transport pathways, in skin could adequately describe the percutaneous absorption of IND, PL and valproic acid. With peptides, a kinetic model including zero-order input rate, first-order permeation rate and first-order degradation rate was able to describe well the steady-state flux of peptides.

SNARE complex zipping as a driving force in the dilation of proteinaceous fusion pores.

The assembly of SNARE proteins into a tight complex has been hypothesized to drive membrane fusion. A model of the initial fusion pore as a proteinaceous channel formed by SNARE proteins places their membrane anchors in separate membranes. This leaves the possibility of a final assembly step that brings the membrane anchors together and drives fusion pore expansion. The present study develops a model for expansion in which the final SNARE complex zipping step drives a transition from a proteinaceous fusion pore to a lipidic fusion pore. An estimate of the energy released upon merger of the helical segments of the SNARE motifs with the helical segments of the membrane anchors indicates that completing the assembly of a few SNARE complexes can overcome the elastic energy that opposes lipid bilayer deformation into a narrow fusion pore. The angle between the helical axes of the membrane anchor and SNARE motif serves as a useful reaction coordinate for this transition. Energy was calculated as a function of this angle, incorporating contributions from membrane bending, SNARE complex assembly, membrane anchor flexing and hydrophobic interactions. The rate of this transition was evaluated as a process of diffusion over the barrier imposed by these combined energies, and the rates estimated were consistent with experimental measurements.

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca2+]i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca2+. Maximal level of Ca2+, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A2 and/or Ca2+/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca2+]i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca2+-sparks and Ca2+-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca2+-microdomain formation, where the “vicious circle” of Ca2+-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca2+-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na+ and Ca2+ (lipid pore).

Light-Induced Permeability Changes in Liposomes Containing Photo-Polymerizable Phospholipids

We have designed a novel class of light-triggerable liposomes prepared from a photo-polymerizable phospholipid DC8,9PC (1,2- bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine). Exposure to UV radiation (254 nm) for 0-45 minutes (25 0C) resulted in photo-polymerization of DC8,9PC in these liposomes and the release of an encapsulated fluorescent dye (calcein). Photopolymerization and permeability changes did not occur from UV-triggered Egg PC/DC8,9PC liposomes. We propose that phase separation and packing of polymerizable lipids in the liposome bilayer are major determinants of photo-polymerization resulting in the formation of local defects and/or lipidic pores in the liposome membrane. Differential Scanning Calorimetry show phase transition peaks at 36.8 0C and 41.6 0C, respectively, in liposomes composed of DPPC:DC8,9PC (9:1 mole ratio) indicating that the reactions occurred while these lipids are in the gel phase (25 0C). Our results indicate that DC8,9PC and DPPC molecules undergo de-mixing in the gel phase. This hypothesis is supported by Molecular Dynamics simulations that indicate separation of DC8,9PC and DPPC in the solid phase lipid bilayer. Cryo-electron microscopic images of the liposomes show major changes in liposome morphology after UV irradiation. When an appropriate tunable photo-sensitizer dye is included in the aqueous compartment of these liposomes, release of contents is triggered by excitation with a laser at the wavelength of the encapsulated dye. Inhibition of release in the presence of oxygen radical scavengers indicate that the mechanism of release involves chemical changes in DC8,9PC unrelated to photo-polymerization. The laser-mediated chemical modifications in DC8,9PC are being analyzed by MS, LC, GC and NMR. We are further developing these liposomes for undergo triggered release of chemotherapeutic agents (e.g. doxorubicin) and are testing their efficacy in vitro and in vivo.

Nanopore Formation in Cells Exposed to Nanosecond Electric Pulses

Chemical and physical insults such as electroporation may compromise the membrane integrity and allow leak ion currents through de-novo formed lipid pores. These pores are thought to short-circuit the membrane and hinder ion channels' physiological function rather than to complement it. However, here we report that brief electric stimuli can trigger formation of membrane pores with specific behaviors that are traditionally considered to be unique for protein ion channels; still other behaviors of these pores distinguish them from both “conventional” electropores and any known ion channels. We found that a single electric shock (600-ns duration, 1 to 5 kV/cm) causes minutes-long increase of membrane electrical conductance due to formation of long-lived, voltage- and current-sensitive, rectifying, cation-selective, asymmetrical pores of nanometer diameter (“nanopores”). Once induced, nanopores oscillate between open and quasi-open (electrically silent) states, followed by either gradual resealing or abrupt breakdown into larger pores, with immediate loss of nanopore-specific behaviors. The formation and extended lifetime of nanopores were verified by non-electrophysiological methods, namely by fluorescent detection of Tl+ uptake and of phosphatidylserine externalization. Apparently, nanopores are not unique to cell stimulation with nanosecond electric pulses, but may form under various physiological and pathological conditions. Nanopores appear adequately equipped for certain functions that are traditionally ascribed to ion channels. Clear distinction between nanopores- and ion channels-mediated currents may be critical for understanding how these currents are controlled.Supported by NIH (NCI) R01CA125482.

Inhibition of Membrane Stretching Prevents Lipid Pore Enlargement in Cells Porated by Nanosecond Electric Pulses (NSEP)

Recent studies established that nsEP cause formation of stable pores in plasma membrane of mammalian cells. Pores formed by a single nsEP of moderate intensity (e.g., one 600-ns pulse at 3-5 kV/cm) were permeable to alkali cations, but not to a larger propidium cation (van der Waals dimensions 1.6 x 1.4 x 0.8 nm), suggesting that the pore diameter does not exceed 1.4 nm. However, higher nsEP amplitudes and/or exposures to multiple pulses could cause minor propidium uptake. Meanwhile, the mechanism of transformation of initial “nanopores” into larger, propidium-permeable pores remains unclear.One mechanism potentially responsible for nanopore enlargement could be membrane stretching caused by osmotic cell swelling following nsEP exposure.In isoosmotic and even hyperosmotic bath media, membrane permeabilization to small inorganic ions leads to equalization of their concentrations inside and outside of cells. However, larger molecules cannot escape the porated cell interior, creating additional osmotic pressure. To prevent nsEP-induced swelling, the bath buffer can be supplemented with a membrane-impermeable compound, which would prevent the increase of intracellular osmolarity.We demonstrated that addition of sucrose (8-40 mM) to the bath buffer decreases or eliminates propidium uptake by nsEP-exposed cells (60-ns, 30 pulses at 30 kV/cm). However, addition of isosmotic amount of NaCl caused little or no protective effect. These data are indicative of the fact that membrane stretching by osmotic cell swelling could indeed be the cause of nanopores enlargement. This hypothesis is being additionally tested to exclude a potentially biasing effect of sucrose and NaCl addition on the efficiency of initial nanopore opening.The work was supported by R01CA125482 from the National Cancer Institute.

Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations

The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted both atomistic and coarse-grained molecular dynamics simulations of dipalmitoyl-phosphatidylcholine (DPPC) bilayers to study the effect of an asymmetric distribution of lipids between the two monolayers on membrane stability. Highly asymmetric lipid bilayers were found to be surprisingly stable within the submicrosecond time span of the simulations. Even the limiting case of a monolayer immersed in water ruptured spontaneously only after at least 20 ns simulation. A thermal shock could destabilize these kinetically trapped states. We also studied mixed systems composed of DPPC and short tail diC(8)PC lipids, showing that the presence of the cone-shaped short tail lipid facilitates the release of tension in the asymmetric systems via formation of a transmembrane pore. Thus, asymmetric area expansion and curvature stress cooperate to yield bilayer disruption. It appears that, although asymmetric area expansion destabilizes the bilayer structure, the activation energy for transmonolayer lipid re-equilibration is increased. Such a large kinetic barrier can be reduced by lipids with positive spontaneous curvature. These effects are important at the onset of bilayer destabilization phenomena, such as lipid pore formation and membrane fusion, and should be considered for the mechanism of induction of such processes by peptides and proteins.

Experimental and theoretical studies of self-organization of complex protein structures

.Virus infection is a multistage process that starts with endocytosis followed by an exit of the viral genetic material into the cell cytoplasm. A lot of proteins participate in the process of endocytosis. At the last stages of endocytosis the membrane neck is represented by a membrane nanotube (NT) with luminal radius of a few nanometers. A set of GTPase dynamines is responsible for the NT fission. After the NT fission, virus-containing vesicle fuses with endosome, which moves to a cell nucleus along microtubules. Subsequent decrease of pH in endosome results in an activation of haemagglutinines (HA). Their conformational rearrangement is followed by self-organization of the protein complex resulting in a formation of fusion pore. The virus RNA exit into the cytoplasm occurs after destruction of M1 protein carcass surrounding nucleoproteins. Apparently, M1 proteins interact with each other as well as with ribonucleoproteins and lipid bilayer in a pHdependent way. Self-organization of proteins in complex structures is mediated by the lipid component of the membranes. In particular, it is shown that proteins in the membrane are collected in extensive aggregates, so-called “islands”, and the part of the membrane in these aggregates is in a liquid-ordered (raft) state. As we suppose, the appearance of rafts can be stipulated by the process of self-organization of protein-lipid systems and involve the mechanism of protein wetting by lipids. A loss of the membrane integrity can be produced by various factors. It always starts with formation of lipid or lipid-protein pores and leads to the cell death. Progressing of the pores can be promoted by an electric field, osmotic stress, or chemical modification of the membrane by the change of lipid composition or formation of lipid-protein structures. Usage of modern methods and approaches has allowed a considerable progress in understanding the virus infection and associated processes of self-organization of complex protein and lipid-protein structures.

The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.

Assembly of the Mitochondrial Apoptosis-induced Channel, MAC

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane alpha-helices in Bax and Bak, mature MAC pores would typically contain approximately 9 monomers and have diameters of 5.5-6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.

Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the "pores" and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25-100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.

Breakdown of Charged Lipid Asymmetry as a Result of Lipidic Pore Formation

Negatively charged lipids are usually located in the inner leaflet of the plasma membrane. Their appearance in the outer leaflet is known to correlate with several physiological and pathological conditions in cells. Understanding how membrane lipids lose their asymmetric transmembrane distribution and achieve their nonrandom distribution in cells is a key challenge in cell biology. Negatively charged lipids do not spontaneously exchange between the two leaflets of a lipid bilayer because the polar headgroups cannot readily cross the hydrophobic membrane interior. We hypothesized that the formation of a transient hydrophilic lipidic pore in the membrane leads to diffusive translocation of negatively charged lipids through the pore to the opposite membrane leaflet. To test this hypothesis, we established a variation of the inner field compensation technique for time resolved measurements of membrane boundary potentials in asymmetric bilayer lipid membranes formed by the Montal-Mueller method. External application of electric fields across the bilayer induced transient conductive states. We observed fast transitions between these different conductance levels, reflecting opening and closing of meta-stable lipidic pores. Comparison of the capacitance minimization potential for different asymmetric membranes before and after pore formation confirmed negatively charged lipids transfer across the bilayer. We also constructed a model governing lipid flow rate based on pore analysis and lipid lateral diffusion rate. Together, our study provides a new tool to monitor loss of membrane asymmetry and our results indicate that lipid transfer can occur through lipidic pores.

Evidence for Lipidic Pores

This study revealed the structure and mechanism of pore formation in membranes by Bax-α5 peptide, a segment from the pore-forming domain of Bax. Bax is an apoptosis regulator protein that forms pores in the outer-mitochondrial membrane to release cytochrome-c. Bax-α5 has been shown to reproduce the pore-forming activity of Bax. Bax-a5 induced pores in multiple bilayers were long-ranged correlated into a periodically ordered lattice and analyzed by X-ray anomalous diffraction. The electron density profile unambiguously shows the Bax-α5 pore is of the toroidal (wormhole) type: the two lipid monolayers merge through the pore. This was the first direct structural evidence for the existence of the long speculated lipidic pores. The molecule mechanism of Bax-α5 pore formation was studied by two experiments: pore formation in individual GUVs exposed to Bax-α5 in solutions and the membrane thinning effect caused by the peptides. Bax-α5 exhibited a sigmoidal concentration dependence similar to antimicrobial peptides we've studied: below a threshold concentration, the peptide only binds to membrane inter surface, causing membrane thinning; when the concentration exceeds a critical value, pore formation is activated. Our results suggest that formation of such lipidic pores is a major mechanism for α-pore-forming peptides and proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Structure Of Complexes Of Helix-5 From Bax With Lipid Membranes

Bax is a proapoptotic protein implicated in the release of cell-death activating factors from the mitochondrial intermembrane space. Although the structure of the membrane-bound forms of Bax is unknown, it has been proposed to form proteolipidic pores. Studies with synthetic lipid vesicles have shown that fragments encompassing helix-5 of Bax retain a membrane permeabilization ability that is similar to that of the full-length protein. Here we report on the structure of peptide-membrane complexes formed by a Bax helix-5 peptide and lipid bilayers. The relative orientation of the peptide and the lipids are determined using site-specific infrared spectroscopy, assisted by isotopic labeling of backbone groups with the 13C=18O probe. The peptide is highly α-helical in all lipid membranes studied, and its orientation reveals different binding modes that depend on the bilayer phase state. In partially fluid POPC bilayers helix-5 of Bax lies almost parallel with respect to the membrane plane, most likely interacting at the level of the interface. However, in gel phase DMPC bilayers the peptide adopts a tilted orientation, which suggests a deeper insertion in the membrane. In turn infrared spectroscopy and X-ray diffraction data show that in some instances the Bax helix-5 peptide influences the phase transition properties of the lipids by increasing membrane fluidity. Taken together, these effects can be related with the membrane perturbation properties of the Bax helix-5 fragment and with its mechanism of action as a molecule inducing the formation of lipidic pores.