Lipid Pore Research Articles

Interaction of the BAK Homodimer with the Membrane

Programmed cell death, or apoptosis is an essential biological process in embryogenesis or in the maintenance of homeostasis in higher eukaryotes. In the mitochondrial apoptotic pathways, the pro-apoptotic Bcl-2 family proteins BAK and BAX, upon activation by death signals, are believed to form large oligomeric pores via homodimer formation in the mitochondrial outer membrane. Through these pores of an unknown structure, many apoptotic factors are released from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. We have determined the topographic locations of the residues in the helices α4, α5 and α6 of BAK in the BAK oligomeric pore using the site-directed spin labeling method and/or the IASD (4-acetamido-4′-((iodoacetyl)amino)stilbene-2,2′disulfonic acid) labeling approach. Accessibility parameters of oxygen and NiEDDA to residues in helix α5 showed that the helix is exposed to the membrane on one side, with the opposite side in tertiary contact. The IASD labeling pattern in helix α5 also revealed the same. These results were consistent with the predictions by the BAX ‘BH3-in-groove homodimer’, a recently reported X-ray crystallographic structure of a dimer of a truncated BAX consisting of helices α2-α5. The membrane immersion-depths of selected residues in helices α4 and α5 indicated that the adsorption of the BAK ‘BH3-in-groove homodimer' to the membrane surface is mediated by the hydrophobic surface of the homodimer. The helix α6 was also adsorbed to the membrane surface, with its N-terminus deeper than the C-terminus. In summary, the data suggest that BAK proteins form a lipidic pore, unlike the channel forming domains of certain bacterial toxins such as diphtheria toxin or colicin molecules. The results provide further insights into the mechanism of formation of the mitochondrial apoptotic pores by BAX or BAK.

Dye Release Experiments with Dextran Loaded Vesicles

Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked to rhodamine or fluorescein are both encapsulated in lipid vesicles to allow quantifying the leakage of each dextran individually from a single sample. For instance, the size evaluation of the lipid pore formed by an antimicrobial peptide has been recently achieved using this protocol (Sani et al., 2013)., 染料释放实验是一种广泛使用的方法来评估膜 - 活性分子和脂质膜之间的相互作用。 特别感兴趣的是通过同时包封不同尺寸的染料（例如用发色团接枝的葡聚糖）来评估脂质双层扰动的程度的能力。 在该测定中，连接到罗丹明或荧光素的葡聚糖都封装在脂质囊泡中，以允许从单个样品单独地量化每个葡聚糖的渗漏。 例如，最近使用该方案获得了由抗微生物肽形成的脂质孔的尺寸评价（Sani等人，2013）。

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K(+)) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K(+) channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K(+) channel inactivation and exhibits a remarkable structural plasticity that correlates to K(+) channel inactivation. The transmembrane helix 1 unwinds when the K(+) channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K(+) ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K(+) channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K(+) channel pore.

Facile Method for Preparing Liposomes by Permeation of Lipid–Alcohol Solutions through Shirasu Porous Glass Membranes

We present a novel and facile approach for preparing monodispersed liposomes simply by permeating lipid–alcohol solutions through Shirasu porous glass (SPG) membranes. This method uses the phenomenon of mixing of two miscible liquids at their interface formed near an SPG membrane surface, which induces spontaneous formation of liposomes. The effects of lipid concentration, pore size of the SPG membrane, and the alcohol used for the lipid solution on the structures and properties of the liposomes are studied. The liposome size is around 120 nm, and the size distribution is narrow, regardless of the lipid concentration, pore size, and alcohol used. However, the alcohol used affects the property of liposomes: An increase in the liposome size is clearly observed in a freezing–thawing treatment when isopropyl alcohol is used as the alcohol, whereas the liposome size is unchanged by the freezing–thawing treatment when ethanol is used.

Lytic and non-lytic permeabilization of cardiolipin-containing lipid bilayers induced by cytochrome C.

The release of cytochrome c (cyt c) from mitochondria is an important early step during cellular apoptosis, however the precise mechanism by which the outer mitochondrial membrane becomes permeable to these proteins is as yet unclear. Inspired by our previous observation of cyt c crossing the membrane barrier of giant unilamellar vesicle model systems, we investigate the interaction of cyt c with cardiolipin (CL)-containing membranes using the innovative droplet bilayer system that permits electrochemical measurements with simultaneous microscopy observation. We find that cyt c can permeabilize CL-containing membranes by induction of lipid pores in a dose-dependent manner, with membrane lysis eventually observed at relatively high (µM) cyt c concentrations due to widespread pore formation in the membrane destabilizing its bilayer structure. Surprisingly, as cyt c concentration is further increased, we find a regime with exceptionally high permeability where a stable membrane barrier is still maintained between droplet compartments. This unusual non-lytic state has a long lifetime (>20 h) and can be reversibly formed by mechanically separating the droplets before reforming the contact area between them. The transitions between behavioural regimes are electrostatically driven, demonstrated by their suppression with increasing ionic concentrations and their dependence on CL composition. While membrane permeability could also be induced by cationic PAMAM dendrimers, the non-lytic, highly permeable membrane state could not be reproduced using these synthetic polymers, indicating that details in the structure of cyt c beyond simply possessing a cationic net charge are important for the emergence of this unconventional membrane state. These unexpected findings may hold significance for the mechanism by which cyt c escapes into the cytosol of cells during apoptosis.

Voltage-Gated Lipid Ion Channels.

Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times.

Cytochrome c causes pore formation in cardiolipin-containing membranes

The release of cytochrome c from mitochondria is a key signaling mechanism in apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanisms remain unclear. Cytochrome c (cyt c) binds to and penetrates lipid structures containing the inner mitochondrial membrane lipid cardiolipin (CL), leading to protein conformational changes and increased peroxidase activity. We describe here a direct visualization of a fluorescent cyt c crossing synthetic, CL-containing membranes in the absence of other proteins. We observed strong binding of cyt c to CL in phospholipid vesicles and bursts of cyt c leakage across the membrane. Passive fluorescent markers such as carboxyfluorescein and a 10-kDa dextran polymer crossed the membrane simultaneously with cyt c, although larger dextrans did not. The data show that these bursts result from the opening of lipid pores formed by the cyt c-CL conjugate. Pore formation and cyt c leakage were significantly reduced in the presence of ATP. We suggest a model, consistent with these findings, in which the formation of toroidal lipid pores is driven by initial cyt c-induced negative spontaneous membrane curvature and subsequent protein unfolding interactions. Our results suggest that the CL-cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis.

Cholesterol favors the emergence of a long-range autocorrelated fluctuation pattern in voltage-induced ionic currents through lipid bilayers

The present paper was aimed at evaluating the effect of cholesterol (CHO) on the voltage-induced lipid pore formation in bilayer membranes through a global characterization of the temporal dynamics of the fluctuation pattern of ion currents. The bilayer model used was black lipid membranes (BLMs) of palmitoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine (POPE:POPC) at a 7:3 molar ratio in the absence (BLM0) or in the presence of 30 (BLM30), 40 (BLM40) or 50(BLM50)mol% of cholesterol with respect to total phospholipids. Electrical current intensities (I) were measured in voltage (ΔV) clamped conditions at ΔV ranging between 0 and ±200mV. The autocorrelation parameter α derived from detrended fluctuation analysis (DFA) on temporal fluctuation patterns of electrical currents allowed discriminating between non-correlated (α=0.5, white noise) and long-range correlated (0.5<α<1) behaviors. The increase in |ΔV| as well as in cholesterol content increased the number of conductance states, the magnitude of conductance level, the capacitance of the bilayers and increased the tendency towards the development of long-range autocorrelated (fractal) processes (0.5<α<1) in lipid channel generation. Experiments were performed above the phase transition temperature of the lipid mixtures, but compositions used predicted a superlattice-like organization. This leads to the conclusion that structural defects other than phase coexistence may promote lipid channel formation under voltage clamped conditions. Furthermore, cholesterol controls the voltage threshold that allows the percolation of channel behavior where isolated channels become an interconnected network.

Insight into the Lytic Mechanism of Antimicrobial Piscidin 1 and 3 using QCM-D

In this work, we focused on piscidin 1 (P1) and 3 (P3), which are twenty-two-residue-long amphipathic, cationic antimicrobial peptides (AMPs) isolated from hybrid striped bass. Extensive work has been reported on characterizing the atomic-level structure of both P1 and P3 in magnetically and mechanically aligned bilayers using solid-state NMR. Here we investigated the mechanism of action of both P1 and P3 with supported lipid bilayers (SLB), using a quartz crystal microbalance with dissipation monitoring (QCM-D). In order to mimic bacterial cell membranes, we selected a 3:1 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Liposomes were deposited onto a QCM-D silicon oxide crystal until SLB formation, then P1 and P3 were introduced into the system and the interaction between the peptide and the lipid system was monitored by changes in frequency (Δf) and energy dissipation (ΔD). Different concentrations (2-20 μM) of the peptide and buffer conditions (pH 6.0 and 7.4) were tested. In particular we observed a Δf increase (∼ mass removal) as we increased the concentration of P1 at pH 7.4, suggesting lipid removal, membrane thinning, or pore formation; while at pH 6.0 the Δf decrease (∼ mass addition) was probably due to the electrostatic interaction between the more cationic peptide and the anionic POPG. For a quantitative assessment of the effects on the SLB, we have obtained the shear viscosity and shear modulus as a function of piscidin type and concentration via a fit of the well-known Voigt model to the data. In all cases, ΔD values were a clear indication of the destabilization of the SLB. Overall, this study, which demonstrates quantitative P1 and P3 disruption of a bacterial membrane mimic, assists in better understanding the mode of action of piscidins and related AMPs.

What Makes You Can Also Break You, Part II: Mitochondrial Permeability Transition Pore Formation by Dimers of the F1FO ATP-Synthase?

What Makes You Can Also Break You, Part II: Mitochondrial Permeability Transition Pore Formation by Dimers of the F1FO ATP-Synthase?

Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients KD,A > 10) for the determination of pore sizes with radii comparable to the Förster radius R0. On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

Transmembrane pore formation by the carboxyl terminus of Bax protein

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.

Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

Mitochondrial lipid pore in the mechanism of glutamate-induced calcium deregulation of brain neurons

The work examines the mechanism of central nerve cell death upon stimulation of brain NMDA receptors with the stimulatory mediator glutamate. A prolonged stimulation of neurons with glutamate is known to result in the disorder of Ca2+ homeostasis and severe mitochondrial depolarization followed by cell death. It has been shown that the overload of mitochondria with Sr2+ leads to the release of the cation, medium alkalization, decrease of membrane potential and mitochondrial swelling, indicating a nonspecific permeabilization of the mitochondrial membrane. The permeabilization, in our opinion, is caused by the activation of Ca2+/Sr2+-dependent phospholipase A2 (PLA2), resulting in the formation of free palmitic and stearic acids in the mitochondrial membrane. These fatty acids bind Ca2+ with high affinity and the process of binding is accompanied by the formation of a transient lipid pore—a phenomenon demonstrated earlier on both artificial and mitochondrial membranes. The inhibitors of PLA2 have been shown to suppress permeabilization of mitochondrial membranes. In the culture of granular cerebellum neurons, the PLA2 inhibitors prolonged the lag of the delayed Sr2+ deregulation and membrane depolarization. On the basis of data obtained on isolated mitochondria and neurons we suppose that the initial stages of glutamate-induced Ca2+ deregulation of neurons are underlain by the opening of lipid pores in brain mitochondria.

Control of Lipidic Pore Dynamics by Aqueous Viscosity

It is experimentally known that the viscosities of aqueous solutions are the dominant source of friction in the dynamics of lipidic pores in large vesicles. Building on an existing model of others in which membrane viscosity is assumed to limit pore dynamics, a new theory is developed that also accounts for aqueous viscosity. The equations that describe pore dynamics leads to a three-stage pattern, as in the prior theory, but the parameters of the new theory agree with experiments. The dissipation of energy resulting from friction between a membrane sliding against the aqueous solution, during changes in pore radius, is derived from fundamental principles of fluid mechanics, and the derived curve fits experimental data without the need to introduce fitting parameters.

A Dynamic Model of Fusion Pores in Lipid Bilayers

Biological membranes remodel and change shape in processes like endocytosis, exocytosis, hemifusion, and the expansion of fusion pores. Since the length scale of these processes is generally quite small, computational models are needed to resolve the time course of the membrane. Over the past few decades, physicists and mathematicians have developed variational methods for studying time dependent changes in materials like lipid membranes. Building on a model of a single lipidic pore in a vesicle membrane, we direct the variational approach to the study of fusion pores. A fusion pore is a toroidal structure which connects two planar bilayers. The variational approach calculates the time dependent shape of the fusion pore--the precise shape of the pore that minimizes the membrane energy can be calculated using the variational method. The shape of the fusion pore is a solution to the equations of motion which include the surface forces induced by the classical Helfrich energy of the membrane. The energetics involved in the fusion pore expansion are determined as a function of the lipid composition and initial conditions. The model is based on a diffusive interface and director field approach which determines the position and energy of the membrane. The diffusive interface specifies the material properties of lipid and water, accounts for the lipid movements, and is the basis for deriving the equations of motion.

Aqueous Viscosity Is the Primary Source of Friction in Lipidic Pore Dynamics

A new theory, to our knowledge, is developed that describes the dynamics of a lipidic pore in a liposome. The equations of the theory capture the experimentally observed three-stage functional form of pore radius over time—stage 1, rapid pore enlargement; stage 2, slow pore shrinkage; and stage 3, rapid pore closure. They also show that lipid flow is kinetically limited by the values of both membrane and aqueous viscosity; therefore, pore evolution is affected by both viscosities. The theory predicts that for a giant liposome, tens of microns in radius, water viscosity dominates over the effects of membrane viscosity. The edge tension of a lipidic pore is calculated by using the theory to quantitatively account for pore kinetics in stage 3, rapid pore closing. This value of edge tension agrees with the value as standardly calculated from the stage of slow pore closure, stage 2. For small, submicron liposomes, membrane viscosity affects pore kinetics, but only if the viscosity of the aqueous solution is comparable to that of distilled water. A first-principle fluid-mechanics calculation of the friction due to aqueous viscosity is in excellent agreement with the friction obtained by applying the new theory to data of previously published experimental results.

The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.

Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry

The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.