Planar Bilayer Membranes Research Articles

Naphthalene Diimide-Based Metallacage as an Artificial Ion Channel for Chloride Ion Transport.

Developing synthetic molecular devices for controlling ion transmembrane transport is a promising research field in supramolecular chemistry. These artificial ion channels provide models to study ion channel diseases and have huge potential for therapeutic applications. Compared with self-assembled ion channels constructed by intermolecular weak interactions between smaller molecules or cyclic compounds, metallacage-based ion channels have well-defined structures and can exist as single components in the phospholipid bilayer. A naphthalene diimide-based artificial chloride ion channel is constructed through efficient subcomponent self-assembly and its selective ion transport activity in large unilamellar vesicles and the planar lipid bilayer membrane by fluorescence and ion-current measurements is investigated. Molecular dynamics simulations and density functional theory calculations show that the metallacage spans the entire phospholipid bilayer as an unimolecular ion transport channel. This channel transports chloride ions across the cell membrane, which disturbs the ion balance of cancer cells and inhibits the growth of cancer cells at low concentrations.

Electric Fields at the Lipid Membrane Interface.

This review presents a comprehensive analysis of electric field distribution at the water-lipid membrane interface in the context of its relationship to various biochemical problems. The main attention is paid to the methodological aspects of bioelectrochemical techniques and quantitative analysis of electrical phenomena caused by the ionization and hydration of the membrane-water interface associated with the phase state of lipids. One of the objectives is to show the unique possibility of controlling changes in the structure of the lipid bilayer initiated by various membrane-active agents that results in electrostatic phenomena at the surface of lipid models of biomembranes-liposomes, planar lipid bilayer membranes (BLMs) and monolayers. A set of complicated experimental facts revealed in different years is analyzed here in order of increasing complexity: from the adsorption of biologically significant inorganic ions and phase rearrangements in the presence of multivalent cations to the adsorption and incorporation of pharmacologically significant compounds into the lipid bilayer, and formation of the layers of macromolecules of different types.

Different effects of two Poloxamers (L61 and F68) on the conductance of bilayer lipid membranes.

The integral conductance of planar lipid bilayer membranes in the presence of two Poloxamers (Pluronics) L61 and F68 with the same lengths of hydrophobic poly(propylene oxide) blocks and the different lengths of hydrophilic poly(ethylene oxide) blocks increases with an increase in the concentration of both Pluronics; however, the shape of the conductance-concentration curves is super linear for L61 and sublinear for F68. In the presence of both Pluronics, rare discrete current jumps are observed against the background of continuous current. At high concentrations, the I-V curves of membranes with both L61 and F68 became nonlinear at sufficiently low voltages but differed significantly. At voltages greater than 50mV, the conductance of membranes with L61 increased sharply and quantized jumps were observed toward higher conductance, which could be interpreted as the appearance of additional pores. On the contrary, the conductance of membranes with F68 decreased and quantized jumps to lower conductance were observed, which could be interpreted as blocking of already existing pores. We attributed the differences in the conductance-concentration and I-V curves of these two Pluronics to their different effects on the dynamics of membrane hydration and, accordingly, on the probability of formation of conducting pores.

Furosemide blocks the anion channel caused by Staphylococcus aureus α-hemolysin

To investigate the effect of furosemide on Staphylococcus aureus α-hemolysin (α-HL) channel in planar lipid bilayers by electrophysiological characterization and molecular docking studies. Planar lipid bilayer membranes were prepared and α-HL (0.07 mg/mL) was added to the standard solution in cis compartment of the experimental chamber. All experiments were performed at room temperature using an Axopatch 200A amplifier in the voltage clamp mode. At pH 7.5, α-HL channels were usually in a high conductance ~4 nS and rarely switch to low conductance states. After the ion channel was incorporated in bilayer membrane, the furosemide was also added to the standard solution to the cis compartment. To docking studies, atomics coordinates for the α-HL heptameric channel was retrieved from PDB ID (7AHL) and the structure of furosemide was removed from the PubChem, their coordinates were built and minimized with Avogadro software. The molecular docking experiments were performed using the Dockthor online. The furosemide inhibited (P<0.05) conductance α-HL channel and it was a voltage-dependent manner. The two best docking solutions and the α-HL channel were evaluated, it was observed the connection mode with the highest affinity of interaction has a greater number of hydrogen bonding. The residues were 113 and 147 that form the remainders of the constriction α-HL channel. In conclusion, furosemide blocks ion currents in the constriction of channel caused by Staphylococcus aureus α-hemolysin.

Preparation of Planar Lipid Bilayer Membrane by Utilizing Bicelles and Its Characterization

Preparation of Planar Lipid Bilayer Membrane by Utilizing Bicelles and Its Characterization

Characterization of Phase Separated Planar Lipid Bilayer Membrane by Fluorescence Ratio Imaging and Scanning Probe Microscope

The lipid membrane forms nanodomains (rafts) and shows heterogeneous properties. These nanodomains relate to significant roles in various cell functions, and thus the analysis of the nanodomains in phase-separated lipid membranes is important to clarify the function and role of the nanodomains. However, the lipid membrane possesses small-sized nanodomains and shows a small height difference between the nanodomains and their surroundings at certain lipid compositions. In addition, nanodomain analysis sometimes requires highly sensitive and expensive apparatus, such as a two-photon microscope. These have prevented the analysis by the conventional fluorescence microscope and by the topography of the scanning probe microscope (SPM), even though these are promising methods in macroscale and microscale analysis, respectively. Therefore, this study aimed to overcome these problems in nanodomain analysis. We successfully demonstrated that solvatochromic dye, LipiORDER, could analyze the phase state of the lipid membrane at the macroscale with low magnification lenses. Furthermore, we could prove that the phase mode of SPM was effective in the visualization of specific nanodomains by properties difference as well as topographic images of SPM. Hence, this combination method successfully gave much information on the phase state at the micro/macro scale, and thus this would be applied to the analysis of heterogeneous lipid membranes.

Why is the SARS-CoV-2 Omicron variant milder?

Why is the SARS-CoV-2 Omicron variant milder?

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.

Historical Perspective of Pore-Forming Activity Studies of Voltage-Dependent Anion Channel (Eukaryotic or Mitochondrial Porin) Since Its Discovery in the 70th of the Last Century.

Eukaryotic porin, also known as Voltage-Dependent Anion Channel (VDAC), is the most frequent protein in the outer membrane of mitochondria that are responsible for cellular respiration. Mitochondria are most likely descendants of strictly aerobic Gram-negative bacteria from the α-proteobacterial lineage. In accordance with the presumed ancestor, mitochondria are surrounded by two membranes. The mitochondrial outer membrane contains besides the eukaryotic porins responsible for its major permeability properties a variety of other not fully identified channels. It encloses also the TOM apparatus together with the sorting mechanism SAM, responsible for the uptake and assembly of many mitochondrial proteins that are encoded in the nucleus and synthesized in the cytoplasm at free ribosomes. The recognition and the study of electrophysiological properties of eukaryotic porin or VDAC started in the late seventies of the last century by a study of Schein et al., who reconstituted the pore from crude extracts of Paramecium mitochondria into planar lipid bilayer membranes. Whereas the literature about structure and function of eukaryotic porins was comparatively rare during the first 10years after the first study, the number of publications started to explode with the first sequencing of human Porin 31HL and the recognition of the important function of eukaryotic porins in mitochondrial metabolism. Many genomes contain more than one gene coding for homologs of eukaryotic porins. More than 100 sequences of eukaryotic porins are known to date. Although the sequence identity between them is relatively low, the polypeptide length and in particular, the electrophysiological characteristics are highly preserved. This means that all eukaryotic porins studied to date are anion selective in the open state. They are voltage-dependent and switch into cation-selective substates at voltages in the physiological relevant range. A major breakthrough was also the elucidation of the 3D structure of the eukaryotic pore, which is formed by 19 β-strands similar to those of bacterial porin channels. The function of the presumed gate an α-helical stretch of 20 amino acids allowed further studies with respect to voltage dependence and function, but its exact role in channel gating is still not fully understood.

Structure and Formation Mechanism of Antimicrobial Peptides Temporin B- and L-Induced Tubular Membrane Protrusion.

Temporins are a family of antimicrobial peptides (AMPs) isolated from frog skin, which are very short, weakly charged, and highly hydrophobic. They execute bactericidal activities in different ways from many other AMPs. This work investigated morphological changes of planar bilayer membranes composed of mixed zwitterionic and anionic phospholipids induced by temporin B and L (TB and TL) using all-atom and coarse-grained molecular dynamics simulations. We found that TB and TL fold to α-helices at the membrane surface and penetrate shallowly into the bilayer. These short AMPs have low propensity to induce membrane pore formation but possess high ability to extract lipids out. At relatively high peptide concentrations, the strong hydrophobicity of TB and TL promotes them to aggregate into clusters on the membrane surface. These aggregates attract a large amount of lipids out of the membrane to release compression induced by other dispersed peptides binding to the membrane. The extruded lipids mix evenly with the peptides in the cluster and form tubule-like protrusions. Certain water molecules follow the movement of lipids, which not only fill the cavities of the protrusion but also assist in maintaining the tubular structures. In contrast, the peptide-free leaflet remains intact. The present results unravel distinctive antimicrobial mechanisms of temporins disturbing membranes.

Extract from the Marine Seaweed Padina pavonica Protects Mitochondrial Biomembranes from Damage by Amyloidogenic Peptides.

The identification of compounds which protect the double-membrane of mitochondrial organelles from disruption by toxic confomers of amyloid proteins may offer a therapeutic strategy to combat human neurodegenerative diseases. Here, we exploited an extract from the marine brown seaweed Padina pavonica (PPE) as a vital source of natural bioactive compounds to protect mitochondrial membranes against insult by oligomeric aggregates of the amyloidogenic proteins amyloid-β (Aβ), α-synuclein (α-syn) and tau, which are currently considered to be major targets for drug discovery in Alzheimer’s disease (AD) and Parkinson’s disease (PD). We show that PPE manifested a significant inhibitory effect against swelling of isolated mitochondria exposed to the amyloid oligomers, and attenuated the release of cytochrome c from the mitochondria. Using cardiolipin-enriched synthetic lipid membranes, we also show that dye leakage from fluorophore-loaded vesicles and formation of channel-like pores in planar bilayer membranes are largely prevented by incubating the oligomeric aggregates with PPE. Lastly, we demonstrate that PPE curtails the ability of Aβ42 and α-syn monomers to self-assemble into larger β-aggregate structures, as well as potently disrupts their respective amyloid fibrils. In conclusion, the mito-protective and anti-aggregator biological activities of Padina pavonica extract may be of therapeutic value in neurodegenerative proteinopathies, such as AD and PD.

The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop

The connector protein, also known as the portal protein, located at the portal vertex in the Phi29 bacteriophage has been found to play a key role in the genome DNA packaging motor. There is a disordered region, composed of 12 sets of 18-residue loops N229–N246, that has been assumed to serve as a “clamp” to retain the DNA within the pressurized capsid when DNA is fully packaged. However, the process remains undefined about how the clamping of DNA occurs and what signal is used to engage the channel loops to clamp the DNA near the end of DNA packaging. In this study, we use the planar lipid bilayer (PLB) membrane technique to study the connector with its loops cleaved. The channel properties are compared with those of the connector with corresponding wild-type loops at different membrane potentials. On the basis of the hypothesis of the Donnan effects in the flashing Brownian ratchet model, we associate the PLB experimental results with the outcomes from the relevant biochemical experiments on the proheads containing the connectors without the loops, which enables us to provide a clear picture about how the DNA clamping occurs. A mathematical relationship between the Donnan potential and the DNA packaging density is established, demonstrating that they are both in essence the same signal that is received and transmitted by the connector to dictate DNA clamping and the termination of DNA packaging. At the end of the study, the PLB technique is proposed as a viral research tool, and its potential use to study the functions of specific domains in a portal protein of the tailed bacteriophages is highlighted.

Rapid fabrication of teflon apertures by controlled high voltage pulses for formation of free standing planar lipid bilayer membrane.

Free standing artificial lipid bilayers are widely used in the study of biological pores. In these types of studies, the free standing planar lipid bilayer is formed over a micron-sized aperture consisting of either polymer such as Polytetrafluoroethylene (PTFE, Teflon) or glass. Teflon is chemically inert, has a low dielectric constant, and has a high electrical resistance which combined allow for obtaining low noise recordings. This study investigates the reproducible generation of micropores in the range of 50-100 microns in diameter in a Teflon film using a high energy discharge set-up. The discharger set-up consists of a microprocessor, a transformer, a voltage regulator, and is controlled by a computer. We compared two approaches for pore creation: single and multi-pulse methods. The results showed that the multi-pulse method produced narrower aperture size distributions and is more convenient for lipid bilayer formation, and thus would have a higher success rate than the single-pulse method. The bilayer stability experiments showed that the lipid bilayer lasts for more than 33h. Finally, as a proof-of-concept, we show that the single and multi-channel electrophysiology experiments were successfully performed with the apertures created by using the mentioned discharger. In conclusion, the described discharger provides reproducible Teflon-pores in a cheap and easy-to-operate manner.

Tamoxifen inhibits the anion channel induced by Staphylococcus aureus α-hemolysin: electrophysiological and docking analysis

To investigate the effects of tamoxifen on Staphylococcus aureus α-hemolysin channel (α-HL) in planar lipid bilayers with electrophysiological characterization and docking studies. Planar lipid bilayer membranes were prepared and α-HL (0.07 mg/mL) was added to the standard solution in cis compartment of the experimental chamber. All experiments were performed at room temperature using an Axopatch 200A amplifier in the voltage clamp mode. At pH 7.5, α-HL channels were usually in a high conductance ~4 nS and rarely switch to low conductance states. After the ion channel was incorporated in bilayer membrane, the tamoxifen was also added to the standard solution to the cis compartment. To docking studies, atomics coordinates for the α-HL heptameric channel was retrieved from PDB ID (7AHL) and the structure of tamoxifen was removed from the Pubchem, their coordinates were built and minimized with Avogadro software. The molecular docking experiments were performed using the Dockthor online portal. The tamoxifen inhibited (P < 0.05) α-HL channel conductance and it was a voltage-dependent manner. The three best docking solutions and the α-HL channel were evaluated, it was observed the connection mode with the highest affinity of interaction has a greater number of types of polar interaction. The residues present interactions of greater energy were 111 and 147 that form the remainders of the constriction in the channel of α-HL. The other conformers were accommodated in a region with more hydrophobic characteristics (valine 149). The mechanism of Staphylococcus aureus α-hemolysin inhibition by tamoxifen was blockade over the constriction of channel.

The Pore-Forming Properties of SsoHel308 Helicase from Saccharolobus solfataricus

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein embedded in rabbit erythrocyte membranes may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a planar artificial bilayer membrane and acts as a transformer. After embedding this enzyme into biolayer lipid membranes, the membrane conductivity is altered. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell. The possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

Modulation of the mitochondrial voltage-dependent anion channel (VDAC) by hydrogen peroxide and its recovery by curcumin.

The Voltage-Dependent Anion Channel (VDAC) plays a vital role in mitochondria-mediated transport of ions and metabolites. It is well established that mitochondria are a site for production of hydrogen peroxide (H2O2). Excess production of H2O2 is toxic to the cell and causes oxidative stress. Therefore, the effect of H2O2 on the single-channel conductance of VDAC was investigated. In vitro bilayer electrophysiology experiments were performed on VDAC isolated from rat brain mitochondria, which consists predominately of the isoform VDAC1. VDAC was treated with H2O2 on a planar bilayer membrane (BLM). The conductance of VDAC increased upon H2O2 treatment, whereas the same concentration of H2O2 was unable to affect the BLM (without protein) over a long period of time. Subsequently, the sequential addition of curcumin to H2O2-treated VDAC reduced the conductance. Experimental results (bilayer electrophysiology) demonstrate the role of curcumin in the restoration of the activity of VDAC affected by H2O2. In silico docking studies enables identification of the probable binding site of H2O2 on VDAC. We further find that the oligomerization of VDAC that results in its increased conductance is an effect of lipid oxidation by H2O2.

Estimation of the parameters of the Smoluchowski equation describing the occurrence of pores in a bilayer lipid membrane under soft poration.

The conductive lipid pores occurring in planar bilayer membranes are known to manifest themselves experimentally as current fluctuations. Reliable recording of such fluctuations during phase transitions, as well as in membranes with various additives (for example, SDS), allows one to determine the characteristics of hypothetical hydrophilic pores, namely, their number, sizes, lifetimes, and duration of time intervals between pores. Because, in contrast with electroporation, the emergence of pores in a membrane does not require high voltages, this process is called soft poration. Studying the characteristics of pores under soft poration allows us to estimate the parameters of the Smoluchowski equation and compare them with the corresponding parameters used to describe electroporation. In this work, the experimental characteristics of current fluctuations in the membrane with the addition of SDS to the bulk solution were used to estimate the parameters of the Smoluchowski equation: the pore edge tension, the energy of the hydrophobic pore/hydrophilic pore barrier, the coefficient of pore diffusion in the radius space, the initial distribution density of the number of pores, and the attempt rate density of the lipids in a membrane. The obtained estimates are close to the parameter values used in studies of electroporation.

Ultra-Stable Freestanding Lipid Membrane Array: Direct Visualization of Dynamic Membrane Remodeling with Cholesterol Transport and Enzymatic Reactions.

Cell membranes actively change their local compositions, serving essential biological processes such as cellular signaling and endocytosis. Although membrane dynamics is vital in the cellular functions, the complexity of natural membranes has made its fundamental understanding and systematic assessment difficult. Here, a powerful artificial membrane system is developed for real-time visualization of the spatiotemporal dynamics of membrane remodeling. Through well-defined air/oil/water interfaces on grid holes, tens of planar lipid bilayer membranes are easily created, and their reproducibility, controllability, and generality are highlighted. The freestanding membranes are large but also highly stable, facilitating direct long-term monitoring of dynamic membrane reconstitution caused by external stimuli. As an example to demonstrate the superiority of this membrane system, the effect of cholesterol trafficking, which significantly affects biophysical properties of cell membranes, is investigated at different membrane compositions. Cholesterol transport into and out of the membranes at different rates causes anomalous lipid arrangements through cholesterol-mediated phase transitions and decomposition, which have never been witnessed before. Furthermore, enzyme-induced membrane dynamics is successfully shown in this platform; sphingomyelinases locally generate asymmetry between two membrane leaflets. This technique is broadly applicable for exploring the membrane heterogeneity under various membrane-based reactions, providing valuable insight into the membrane dynamics.

Regulation of Single-Channel Conductance of Voltage-Dependent Anion Channel by Mercuric Chloride in a Planar Lipid Bilayer.

The existence of mercury in various forms, e.g., elemental, organic, and inorganic has been known for decades. In any of these forms, it is poisonous to metabolism. In this, an investigation about the effect of the inorganic form of mercury, i.e., mercuric chloride (HgCl2) to the mitochondrial voltage-dependent anion channel (VDAC), has been done after isolation from the cardiac and brain tissues of Wistar rats. In vitro electrophysiology experiments were performed in Cardiolipin planar lipid bilayer membrane (BLM) to study the change in the conductance, selectivity, and gating charge of VDAC post HgCl2 treatment. A reduction in mean conductance of VDAC from 4.3 ± 0.18 to 1.66 ± 0.11 nS was observed. Further, the Gating charge calculated before (± 3.5) and after HgCl2 treatment (± 2.3) showed significant difference. Later, VDAC's behavior was studied at different concentrations of HgCl2 ranging from 0.1μM to 1mM. The Inhibitory concentration (IC50) was calculated from the linear regression plot. The IC50 was found to be 488.1μM. In the asymmetrical HgCl2 (5:1), a permeability ratio of cation to anion was found to be 4.2. It is interpreted that VDAC functioning is affected due to the application of 4mM HgCl2 and a reduction in the conductance, gating charge, and permeability of VDAC was detected. The results provide clues to HgCl2-induced toxicity mediated through VDAC in the Cardiolipin BLM.

Enhancement of direct membrane penetration of arginine-rich peptides by polyproline II helix structure

The left-handed, extended polyproline II (PPII) helix is a unique secondary structure which potently modulates peptide/protein functions through its constraint conformation. To investigate the effect of PPII helix on the direct cell membrane penetration of arginine-rich peptides, we designed a polyproline-containing arginine-rich peptide P9R7W (PPPPPPPPPRRRRRRRW) by introducing nine proline residues into a linear R7W (RRRRRRRW) peptide. Circular dichroism spectroscopy showed that P9R7W has the PPII helix structure in solution whereas R7W is predominantly in random coil structure. Tryptophan fluorescence measurements demonstrated that P9R7W binds to negatively charged lipid vesicles with similar affinity to R7W, in which there was no significant change in the PPII helix structure. Flow cytometry and confocal laser scanning microscopy analyses showed that P9R7W has an ability to penetrate into CHO-K1 cells more efficiently compared to R7W with no cytotoxicity. Consistently, a channel current analysis unveiled that P9R7W penetrates planar lipid bilayer membranes more efficiently than R7W without significant membrane perturbation. Our results indicate that the PPII helix structure can enhance the membrane penetration efficiency of arginine-rich peptides without lipid membrane perturbation.