Planar Bilayer Membranes Research Articles

Disruption of model cell membranes by carbon nanotubes

Carbon nanotubes (CNTs) have one of the highest production volumes among carbonaceous engineered nanoparticles (ENPs) worldwide and are have potential uses in applications including biomedicine, nanocomposites, and energy conversion. However, CNTs possible widespread usage and associated likelihood for biological exposures have driven concerns regarding their nanotoxicity and ecological impact. In this work, we probe the responses of planar suspended lipid bilayer membranes, used as model cell membranes, to functionalized multi-walled carbon nanotubes (MWCNT), CdSe/ZnS quantum dots, and a control organic compound, melittin, using an electrophysiological measurement platform. The electrophysiological measurements show that MWCNTs in a concentration range of 1.6–12ppm disrupt lipid membranes by inducing significant transmembrane current fluxes, which suggest that MWCNTs insert and traverse the lipid bilayer membrane, forming transmembrane carbon nanotubes channels that allow the transport of ions. This paper demonstrates a direct measurement of ion migration across lipid bilayers induced by CNTs. Electrophysiological measurements can provide unique insights into the lipid bilayer–ENPs interactions and have the potential to serve as a preliminary screening tool for nanotoxicity.

Insertion of an Anionic Analogue of the Antimicrobial Peptide PGLa in Lipid Architectures Including S-Layer Supported Lipid Bilayers

The membrane – peptide insertion behavior of an artificial antimicrobial peptide analogue in liposomes, planar free-standing bilayer and planar lipid membranes supported by a crystalline bacterial surface layer, termed S-layer, was investigated. The template for this peptide was peptidyl-glycine-leucine-carboxyamide (PGLa) where all lysine residues were replaced by glutamic acid resulting in a negatively charged analogue termed PGLa(-). Zeta potential measurements and calcein release experiments on liposomes revealed that the insertion of PGLa(-) can be compared to that of native antimicrobial peptides. Patch clamp recordings on free-standing lipid membranes provided evidence of pore formation at a lipid to peptide ratio (L/P) of 1600 with a single pore conductance of 25 pS. However, also a lower conductance at a high L/P (3200) was observed which might be explained by membrane disordering effects caused by PGLa(-) interaction. In line with other studies on the action of membrane active peptides, the rupture of the lipid membrane was strongly influenced by the peptide concentration. S-layer supported lipid membranes were utilized to perform combined surface-sensitive (quartz crystal microbalance with dissipation measurements) and electrical (impedance spectroscopy) measurements. These data evidenced not only the attachment and/or insertion of PGLa(-) in the supported lipid membrane but also indicated toroidal pore formation in a concentration dependent fashion. Hence, S-layer supported lipid membranes constitute a promising platform for studying the interaction and insertion of antimicrobial peptides. Keywords: Antimicrobial peptide, electrochemical quartz crystal microbalance with dissipation monitoring, model lipid membranes, nanobiotechnology, patch-clamp, peptidyl-glycine-leucine-carboxyamide, S-layer supported bilayer.

Cationic Dednrimers as a New Group of Channel-Blocking Antitoxins

Binary pore-forming bacterial protein toxins, including anthrax, are among Nature's most potent biological weapons. Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF), which interact at the surface of mammalian cells to form toxic complexes. LF and EF are enzymes that target substrates within the cytosol; PA provides a heptameric pore to facilitate LF and EF transport into the cytosol. In the present study, we evaluate a series of positively charged poly(amido amine) (PAMAM) dendrimers with the goal of identifying potent inhibitors of anthrax toxin. We show that all tested cationic dendrimers block PA63 channels. However, size of the dendrimers increases with the generation number, which limits ability of the large multi-charged dendrimer to enter the channels. We identify optimal size and charge characteristics of the blockers testing the G0 - G8 generations of PAMAM dendrimers against the single PA63 using the high-resolution single-channel measurements in planar bilayer membranes. Besides, we investigate the kinetics of the PAMAM dendrimers - pore binding/dissociation reactions at different voltages and salt concentrations.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Compositional interface dynamics within symmetric and asymmetric planar lipid bilayer membranes

Compositional domains within multicomponent lipid bilayer membranes are believed to facilitate many important cellular processes. In this work, we first derive the general equations that describe the dynamics of compositional domains within planar membranes with asymmetry in leaflet properties and in the presence of a thermodynamic coupling between the leaflets. These equations are then employed to develop analytical solutions for the dynamics of the recurrence of registration for circular domains in the case of weak coupling. In addition, a closed-form expression for the decay rate of interface fluctuations, when only one leaflet supports compositional domains, is derived.

Formation of Planar Bilayer Membranes on Solid Supports Using Peptide Gemini Surfactants

Abstract Peptide-based gemini surfactants, called PG-surfactants, consisting of a tri- or tetra-Asp peptide core and acetylated dodecylamine-conjugated Cys residues at both the N- and C-terminal ends (D3C12 and D4C12), form a stable planar bilayer membrane on a solid support. From the DLS and TEM analyses, D3C12 and D4C12 have a tendency to form bilayer morphologies in a buffer solution, which reflects their tendency to form a stable planar bilayer membrane.

Substitution of ether linkage for ester bond in phospholipids increases permeability of bilayer lipid membrane for SkQ1-type penetrating cations

Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed.

Activation of the p66Shc pathway in mitochondria-related pathologies

have a channel activity [6], mitochondria from ΔPOR1 strain imported significant amount of tRK1 in presence of cytb2-DHFR. Experiments with vesicles prepared from outer membrane of wild type and ΔPOR1,2 mitochondria confirmed the essential role of Por2 protein in tRK1 import. Finally, first time isolated homogenous Por2 was reconstituted into small unilamellar vesicles and planar phospholipid bilayer membranes. With proteoliposomes it was shown that Por2 provides transport of carbohydrates and nucleotides across the membrane with the same kinetic as for Por1 and on bilayer system cation selective channels were registered with conductivity about 2 nS and closure with voltage above 30 mV in pH dependent manner. Physiological role of Por2 and downregulation mechanism of its channel activity in mitochondrial outer membrane are discussed. An understanding of the fine mechanisms of tRNA import from cytosol could help us to improve delivery of “therapeutic” RNA molecules into mitochondria to cure at themolecular level the negative effects of mutations of tRNA genes coded by the mitochondrial DNA causes large number of human diseases, such as Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) or Myoclonic Epilepsy with Ragged Red Fibers (MERRF) [7].

Expression, Purification and Functional Reconstitution of Slack Sodium-Activated Potassium Channels

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

Study of Nucleotide Binding to the Uncoupling Protein 1 using Atomic Force Microscopy

A tight proton transport regulation in the inner mitochondrial membrane is crucial for physiological processes such as ATP synthesis or non-shivering thermogenesis in the presence of uncoupling protein-1 (UCP1). Several hypotheses explaining the inhibitory effect of ATP on UCP were proposed, however, structural details are missing so far. Here we use the topographic and recognition (TREC) mode of an atomic force microscope to visualize UCP1 re-constituted into lipid bilayers (Beck, 2006) and to analyze the ATP-protein interaction on the single molecule level (Wildling, 2011). The comparison of recognition patterns obtained with anti-UCP antibody and ATP led to the conclusion that the ATP binding site can be accessed from both membrane sides. Using cantilever tips with different crosslinker lengths, we determined the location of the nucleotide binding site inside the membrane with 1 A precision. In the absence of the crystallographic structure for UCP1, these data provide valuable insight into the nucleotide binding mechanism.ReferencesBeck, V., Jaburek, M., Jezek, P., Pohl, E.E. (2006). A new automated technique for the reconstitution of hydrophobic proteins in planar bilayer membranes. Reconstitution of recombinant uncoupling protein 1. Biochim. Biophys. Acta (Bioenergetics), 1757(5-6):474-9.Wildling, L., Unterauer, B., Zhu, R., Rupprecht, A., Haselgruebler, T., Rankl, C., Ebner, A., Vater, D., Pollheimer, P., Pohl, E.E., Hinterdorfer, P., Gruber, H. J. (2011) Linking of sensor molecules with amino groups to amino-functionalized AFM tips. Bioconjugate Chem. 22, 1239-1248.

Comprehensive analysis of compositional interface fluctuations in planar lipid bilayer membranes

In this paper we present a comprehensive analysis of line tension-driven compositional interface fluctuations in planar lipid bilayer membranes. Our starting point is the advective Cahn-Hilliard equation for the local lipid composition in symmetric membranes, which explicitly incorporates both advective and diffusive lipid transport processes, and which is coupled to the continuum hydrodynamic equations governing the flow behavior of the membrane and surrounding solvent with finite subphase thickness. In order to extract the interface dynamics from the continuum phase-field formalism, we first derive the appropriate sharp-interface limit equations. We then carry out a linear perturbation analysis for the relaxational dynamics of small-amplitude sinusoidal interface fluctuations to yield the general dispersion relation ω(k) as a function of perturbation wave number k. The resulting expression incorporates the effects of diffusive and advective lipid transport processes within the membrane, viscous or viscoelastic membrane properties, coupling between membrane and solvent, and inertial effects within the membrane and solvent. It is shown that previously considered scenarios naturally emerge as limiting cases of the general result. Furthermore, we discuss two additional scenarios amenable to analysis, one in which the inertia of the solvent is relevant, and another one in which the membrane displays significant viscoelastic properties. Finally, we numerically evaluate the general dispersion relation for three representative model membrane systems.

Masking apertures enabling automation and solution exchange in sessile droplet lipid bilayers

Reconstitution of ion channels and transmembrane proteins in planar lipid bilayer membranes allow for their scientific study in highly controlled environments. Recent work with lipid bilayers formed from mechanically joined monolayers has shown their potential for wider technological application, including automation and parallelization. However, bilayer areas are highly sensitive to variations in mechanical position and the bilayers themselves cannot withstand significant perfusion of adjacent solutions. Toward this end, here we describe use of an aperture that masks the monolayer contact area, enabling formation of highly consistent bilayer areas and significantly reducing their variation with changes in relative position of the monolayers. Further, use of the aperture enables flow of solution adjacent to the bilayer without rupture or significant change in bilayer area. The device design is scalable and compatible with SBS standard instrumentation and automation technology, potentially enabling its use for rapid, parallel automated measurements of ion channels for large scale scientific studies and pharmaceutical screening.

Generation of artificial channels by multimerization of β-strands from natural porin

General diffusion porins are passive transmembrane channels. We have explored the possibility to create artificial nanopores starting from natural β-barrel structures. Structural elements of bacterial porins were used to build a series of artificial nanopores. The basic module was selected by multi-alignment of general diffusion porins. The sequence corresponded to a highly conserved motif containing two β-strands, which was obtained from Escherichia coli OmpF. Dimeric to octameric repeats were obtained through cDNA recombinant technology. The hexameric repeat was used to test its properties. This protein was expressed, purified and reconstituted in the planar bilayer membranes. It was able to form channels in membranes with a conductance of 300 pS in 150 mm KCl and did not show any relevant voltage-dependence.

A kinetic analysis of protein transport through the anthrax toxin channel

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell’s endosomal membrane, disrupting cellular homeostasis. (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LFN, the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LFN molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LFNs. In this paper, we show both in macroscopic and single-channel experiments that even with only one LFN bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LFN bound. We also present a simple electrodiffusion model of translocation in which LFN is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA63)7, in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA63)7 incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LFN (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LFN adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LFN in the normal direction but also can, at least partially, translocate LFN in the opposite direction.

Voltage-Dependent Membrane Insertion and Translocation of α-Synuclein: Liposome Studies

Alpha-synuclein (αSyn), a 140 amino acid protein enriched in neuronal tissue, is implicated in the pathogenesis of Parkinson's Disease (PD), one of the most common neurodegenerative disorders. The membrane appears to be a potential target of αSyn. Studies with oligomeric or aggregated “protofibrillar” or amyloid forms of αSyn, in which β-structure is dominant, have been shown to permeabilize membranes in cell-free systems.1,2 A monomeric αSyn preparation, which could allow determination of a defined structure in membrane provided an alternative perspective.3 αSyn, disordered in solution, assumes a predominately α-helical conformation when bound to liposomes with an anionic surface and high degree of curvature.3,4 Using cysteine scanning mutagenesis to probe the membrane interaction of different regions of αSyn, the interaction of monomeric αSyn with membranes has been studied, thus far with mutants that span the polypeptide, from Val3 to Ala124. Two membrane systems, planar bilayer membranes and liposomes, have utilized an array of tagged cysteines to determine voltage-dependent topology of αSyn in membranes. Voltage-dependent membrane insertion into, and translocation across, liposome anionic phospholipid membranes, was studied through the response of the fluorescence probe 7-nitro-2,1-benzoxadiazole (NBD), covalently attached to individual Cys residues, to a potassium diffusion potential generated by addition of valinomycin to K+-loaded liposomes. Translocation was assessed through quenching of NBD fluorescence by entrapped bromide. A map of the voltage-dependent membrane topology of αSyn was obtained, in response to a potential of −118 mV, in which a segment of αSyn containing Val48Cys is translocated. In liposomes and planar bilayer membranes, translocation with the PD familial mutant E46K was greater than that of wild type. (Support: Michael J. Fox Foundation for Parkinson's Research).

Effect of Antimicrobial Peptide‐Amide: Indolicidin on Biological Membranes

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity is intensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here an in situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membrane of dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependent interaction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purple membrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the border of membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool in the study of indolicidin-membrane interaction.

Multivalent ions control the transport through lysenin channels

We report the effect of different ions on the conducting properties of lysenin channels inserted into planar lipid bilayer membranes. Our observations indicated that multivalent ions inhibited the lysenin channels conductance in a concentration dependent manner. The analysis performed on single channels revealed that multivalent ions induced reversible sub-conducting or closed states depending on the ionic charge and size. Good agreement is reported between experimental results and a theoretical model that is proposed to describe the interaction between divalent ions and lysenin channels as a simple isothermal absorption process.

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

Electrophysiological Method for Quantification of the Number of phi29 DNA Packaging Nanopore in Planar Bilayer Membrane

Bacterial virus phi29 uses one of the strongest DNA packaging nanomotors to package its micron-length genomes into a pro-capsid. After re-engineering, whether the DNA nano-motor can be used to pump drugs, DNA, RNA or other therapeutic molecules into specifically targeted cells represent a great challenge in nanomedicine. We have recently successfully embedded the connector, a core component of phi29 DNA package motor, into a planar bilayer membrane (BLM). Under an electric field, double-stranded DNA translocated through the connector channel.The application of phi29 connector array as a stochastic sensor requires knowledge of the number of channels on each membrane. We herein report a method for precise counting of the number of channels on each membrane by electrophysiological approach. Generally, the number of channels is determined by the conductance of total channels and conductance per single channel. Using a derived empirical equation, we can calculate the conductance per single channel at any salt concentration using conductivity of respective conducting buffer. The total conductance of total channels can be measured by ionic current through all the channels under an applied ramp voltage. Comparison of calculated and true values established this as a feasible, reliable and reproducible approach.

Apparent fractal distribution of open durations in cyclodextrin-based ion channels

Cyclodextrin-based ion channels are readily prepared via a "click" reaction and exhibit a range of membrane activities consistent with the formation of ion channels in planar bilayer membranes. The durations of irregular and transient conductance events appear to follow a power-law scaling.