Artificial Lipid Bilayers Research Articles

Hyperici herba extract interaction with artificial lipid bilayers.

Hyperici herba (Hyp) is the aerial part collected during the flowering period from the well-known herb, Hypericum perforatum. Black lipid membrane experiments were performed to investigate the effect of the ethanolic Hyp extract on the electrical properties (capacitance and conductance) of artificial lipid bilayers. Hyp extract (1-10 microg mL(-1)) induced a concentration-dependent increase of both specific transmembrane capacitance and conductance in phosphatidylcholine (PC) membranes. The effect on conductance was enhanced when the Hyp extract (3 microg mL(-1)) was present on both sides of the membrane (Gm=77.89 +/- 8.81 nS cm(-2), n=5) compared with single-sided application (Gm=36.48 +/- 2.41 nS cm(-2), n=5). In bilayers containing PC and phosphatidylserine (PS), PC:PS, the Hyp extract effect was greater than on pure PC bilayers, although the surface charge was not the determining factor of this enhanced activity. Adding cholesterol to the PC:PS mixture reverted the conductance increase induced by the Hyp extract in a dose-dependent manner. The specific pattern of the Hyp extract interaction with lipid bilayers has possible consequences concerning its absorption and bioavailability, as well as its pharmacodynamic effects on neuronal excitability.

Structure of human CLIC3 at 2 Å resolution

Structure of human CLIC3 at 2 A resolution D. R. Littler,* L. J. Brown, S. N. Breit, A. Perrakis, and P. M. G. Curmi 1 Department of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands 2 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia 3 St Vincent’s Centre for Applied Medical research, St Vincent’s Hospital, Sydney, New South Wales 2010, Australia 4 School of Physics, University of New South Wales, Sydney, New South Wales 2052, Australia

Automated lipid bilayer and ion channel measurement platform

Ion channels and transmembrane proteins play key roles in a wide range of physiological processes. Engineered ion channels have been explored as highly sensitive single molecule sensors. Scientific and sensing measurements of ion channel conductance often utilize artificial lipid bilayers, which have shortcomings limiting their application. We describe a fully automated lipid bilayer formation system that integrates the measurement electronics within the fluidic controls. Unattended operation of this system resulted in highly reproducible automatic bilayer formation and ion channel measurement over dozens of consecutive trials. The fully automated, closed-loop control algorithm enabled autonomous operation of the platform, a step toward applications of ion channel measurements for remote sensing and pharmacological studies requiring minimal operator involvement.

Engineering Lipid Bilayer Platforms for High Throughput Cell-Free Electrophysiology

Ion channels are crucial physiologically, involved in nearly all biological processes. Their many roles make them important drug targets as well as targets to avoid, as for drug safety screening (e.g. the hERG channel). Patch clamp provides the highest quality measurements of ion channel function and is used by the pharmaceutical industry to test drug interactions with ion channels, but has poor cost and throughput performance. As an alternative ion channel measurement platform, artificial lipid bilayers are well established to provide a highly controllable environment capable of measurement at the single molecule level, although they also suffer from their own technological shortcomings. Previously, we developed an artificial lipid bilayer platform which is capable of shipping and indefinite storage. Here we present the continued technological development of this system which has resulted in faster bilayer formation times and the ability to package, store, and ship ion channels with the bilayer chip. We have also begun to integrate array chip designs with automated and high throughput solution manipulation and ion channel measurement hardware, advancing this platform closer to operator-free involvement and low cost high throughput cell-free electrophysiology.

0610 人工脂質二重膜に再構成したタンパク質モータプレスチンの原子間力顕微鏡を用いた構造解析(GS6:細胞と微小組織)

0610 人工脂質二重膜に再構成したタンパク質モータプレスチンの原子間力顕微鏡を用いた構造解析(GS6:細胞と微小組織)

Single channel properties of lysenin measured in artificial lipid bilayers and their applications to biomolecule detection

Single channel currents of lysenin were measured using artificial lipid bilayers formed on a glass micropipette tip. The single channel conductance for KCl, NaCl, CaCl2, and Trimethylammonium-Cl were 474 ± 87, 537 ± 66, 210 ± 14, and 274 ± 10 pS, respectively, while the permeability ratio PNa/PCl was 5.8. By adding poly(deoxy adenine) or poly(L-lysine) to one side of the bilayer, channel currents were influenced when membrane voltages were applied to pass the charged molecules through the channel pores. Current inhibition process was concentration-dependent with applied DNA. As the current fluctuations of α-hemolysin channels is often cited as the detector in a molecular sensor, these results suggest that by monitoring channel current changes, the lysenin channel has possibilities to detect interactions between it and certain biomolecules by its current fluctuations.

Transport of Cephalosporin Antibiotics Across the Outer Membrane

Antibiotic resistance is emerging in Gram-negative bacteria worldwide. The outer membrane of Gram-negative bacteria is a formidable selective barrier, and a major factor in broad-spectrum antibiotic resistance. Influx of antibiotics into the Periplasm of bacteria is facilitated by proteins that form channels in the outer membrane. We studied the influx of several cephalosporin antibiotics through the major Escherichia coli porins OmpF and OmpC.Conductance measurements through purified single porins reconstituted in artificial lipid bilayers allowed us to count the passage of single antibiotic molecules. Statistical analysis of transport events yields the kinetic parameters at the single molecular level. Fluorescence steady-state measurements were used to quantify the interaction between the antibiotics and the porin channels and verify the calculation of translocation kinetics. For the first time, we have been able to characterize facilitated translocation of several β-lactams through OmpC (most expressed porin in vivo) and quantified the distinguishable permeation properties of ceftriaxone, ceftazidime and cefpirome through both outer membrane porins OmpF and OmpC - concluding a stronger interaction with OmpF than OmpC for all three cephalosporins -especially ceftazidime-.Our approach may be of great benefit to the understanding of porin-drug interaction at the molecular scale and may contribute to the rational design of more efficient antibiotics.

Functional Properties of SR Cl- and K+ Channels during Postnatal Development of Cardiac Muscle

In adult (AD) heart, the sarcoplasmic reticulum (SR) contains Cl- & K+ channels presumably involved in controlling RyR-mediated SR Ca2+ release. These channels provide a countercurrent mechanism that attenuates the drop in Ca2+ driving force across the SR membrane, thereby preventing early termination of Ca2+ release. We showed that in newborn (NB), Ca2+ sparks occur with similar frequency than in AD but have shorter duration and smaller amplitude, implying an early termination of Ca2+ release. Although the functional properties of SR Cl- & K+ channels have been thoroughly described in AD, little is known about their presence and their role in NB. Consequently, we first tested the hypothesis that the early termination of Ca2+ release in NB coincides with absence/low density of SR Cl- & K+ channels at this stage. To this end, the heavy microsomal fraction was obtained from 5-days-old NB and AD rat hearts and SR Cl- & K+ channels were reconstituted into artificial planar lipid bilayers. Our results indicate that Cl- & K+ channels can be reconstituted from NB heavy SR microsomes with a similar success rate (number of SR channel incorporations / total number of bilayers) than in AD (∼0.2 for Cl- channels & ∼0.1 for K+ channels). Thus, an alternative mechanism would imply that in NB, smaller counterion fluxes result from different functional properties of SR Cl- & K+ channels. This assumption was tested by measuring their unitary conductance, open probability, and voltage dependence. The results in NB channels revealed no significant differences in any of these parameters in comparison to AD. Thus, we concluded that SR Cl- & K+ channels do not contribute to the developmental changes of Ca2+ release in NB cardiomyocytes. Supported by AHA-0655656Z to RMA.

Effect of Dipole Modifying Agents on the Surfactin Induced Conductance of Planar Lipid Bilayers

We studied effects of compounds changing membrane dipole potential on membrane conductance induced by antimicrobial lipopeptide surfactin. Surfactin added on both sides of an artificial lipid bilayer from diphytanoyl phosphocholine in 1 M KCl (pH 6.5) produces an increase of the membrane conductance as a result of ion channel formation. Increasing a membrane dipole potential adding RH 421 to the bilayer bathing solution (10 µM , both sides) leads to ∼40 times increase of a steady-state conductance. At the same time, addition of phloretin (20 µM), known to decrease the dipole potential, results in decrease of the surfactin-induced conductance by ∼30 times. We note, that the effects of dipole modifiers on the surfactin-induced membrane conductance are clearly opposite to the effects observed with the same modifiers in case of syringomycin E-induced conductance of lipid bilayers [Ostroumova et al., Langmuir, 2008]. As we suggested earlier, the influence of dipole modifiers on syringomycin activity may be related to a promotion/retardation of a movement of positively charged syringomycin molecules in the direction of membrane hydrocarbon core. In contrast to syringomycin, surfactin is negatively charged. Hence, one can expect an inversion of the effect of dipole modifiers in case of surfactin. The obtained results are in agreement with the model proposed in [Ostroumova et al., Langmuir, 2008].The study was supported in part by the Russian Fund for Basic Research (09-04-48860), the State Program of Molecular and Cell Biology, the grant of St.-Petersburg Administration for young scientists, and the State contract (FAE Π-1372).

A microdroplet-based shift register

A microfluidic device is presented for the serial formation, storage and retrieval of water microdroplets in oil. The principle of operation is similar to that of an electronic shift register. Droplets, considered as units of information, can be arrayed and serially shifted within the device, allowing the controllable positioning of the emulsions and the creation of interfaces between drops. Using this passive system, by exploiting the balance between hydrodynamic pressure and surface tension across a drop due to the device design, droplet networks can be readily arrayed in a series of elements and cascaded within the microchannels in an automatable and high throughput fashion. The results showed the suitability of the system to be used for the formation of artificial lipid bilayers and for the study of biological dynamic processes based on the diffusion of molecules through interfaces.

Integrating carbon nanotubes and lipid bilayer for biosensing

Membrane proteins, which are the target of most drugs, are implicated in many critical cellular functions such as signal transduction, bioelectricity, exocytosis and endocytosis. Therefore, developing techniques to investigate the functions of membrane proteins is obviously important. Here, we have developed a novel system by integrating artificial lipid bilayer (biomimetic membrane) with single-walled carbon nanotube networks (SWNT-net) based field-effect transistor (FET), and demonstrated that such hybrid nanoelectronic biosensors can specifically and electronically detect the presence and dynamic activities of ionophores (specifically, gramicidin and calcimycin) in their native lipid environment. This technique can potentially be used to examine other membrane proteins (e.g. ligand-gated ion channels, receptors, membrane insertion toxins, and antibacterial peptides) for the purposes of biosensing, fundamental studies, or high throughput drug screening.

Visualization of cargo concentration by COPII minimal machinery in a planar lipid membrane

Selective protein export from the endoplasmic reticulum is mediated by COPII vesicles. Here, we investigated the dynamics of fluorescently labelled cargo and non-cargo proteins during COPII vesicle formation using single-molecule microscopy combined with an artificial planar lipid bilayer. Single-molecule analysis showed that the Sar1p-Sec23/24p-cargo complex, but not the Sar1p-Sec23/24p complex, undergoes partial dimerization before Sec13/31p recruitment. On addition of a complete COPII mixture, cargo molecules start to assemble into fluorescent spots and clusters followed by vesicle release from the planar membrane. We show that continuous GTPase cycles of Sar1p facilitate cargo concentration into COPII vesicle buds, and at the same time, non-cargo proteins are excluded from cargo clusters. We propose that the minimal set of COPII components is required not only to concentrate cargo molecules, but also to mediate exclusion of non-cargo proteins from the COPII vesicles.

Rapid fabrication of Teflon micropores for artificial lipid bilayer formation

A number of recent studies have dealt with the development of biosensors using single-channel recording with an artificial lipid bilayer. However, the fragility of these bilayers and current noise present serious problems in their application towards biosensor development. To address this problem, many experimental investigations employing micropores in the formation of lipid bilayers have been reported. In this work, we present a method for the fabrication of micropores using commercially available low-melting Teflon film and a heated tip. This method allowed for the rapid (in a few seconds) and reproducible fabrication of micropores 2–3μm in diameter. We employed a single-channel recording using a gramicidin channel and confirmed that the bilayer membrane can form on micropores by the painting method. The bilayer formed is stable under high voltage (+1000mV). Fabricated micropores possess a conical shape with sharp edges, features which facilitated the formation of artificial lipid bilayers which could be utilized for low-noise and high voltage recording due to decreased access resistance and increased bilayer stability. These advantages promise to improve the performance of artificial lipid bilayers when employed in the development of flexible biosensors.

The proximity between C‐termini of dimeric vacuolar H+‐pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique

Vacuolar H(+)-translocating inorganic pyrophosphatase [vacuolar H(+)-pyrophosphatase (V-PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PP(i) hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function, although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton-translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 +/- 0.3 nm and 26.3 +/- 4.7 nm, whereas those of the luminal protrusions were 1.2 +/- 0.1 nm and 21.7 +/- 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6xHis tags on C-terminal ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 2.2 +/- 1.4 nm. Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.

Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles

To explore mechanisms of nanoparticle interactions with and trafficking across lung alveolar epithelium, we utilized primary rat alveolar epithelial cell monolayers (RAECMs) and an artificial lipid bilayer on filter model (ALBF). Trafficking rates of fluorescently labeled polystyrene nanoparticles (PNPs; 20 and 100 nm, carboxylate (negatively charged) or amidine (positively charged)-modified) in the apical-to-basolateral direction under various experimental conditions were measured. Using confocal laser scanning microscopy, we investigated PNP colocalization with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, and wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from RAECMs, and trafficking of (22)Na and (14)C-mannitol across ALBF, were measured in the presence and absence of PNPs. Results showed that trafficking of positively charged PNPs was 20-40 times that of negatively charged PNPs across both RAECMs and ALBF, whereas translocation of PNPs across RAECMs was 2-3 times faster than that across ALBF. Trafficking rates of PNPs across RAECMs did not change in the presence of EGTA (which decreased transepithelial electrical resistance to zero) or inhibitors of endocytosis. Confocal laser scanning microscopy revealed no intracellular colocalization of PNPs with early endosome antigen-1, caveolin-1, clathrin heavy chain, cholera toxin B, or wheat germ agglutinin. Leakage of 5-carboxyfluorescein diacetate from alveolar epithelial cells, and sodium ion and mannitol flux across ALBF, were not different in the presence or absence of PNPs. These data indicate that PNPs translocate primarily transcellularly across RAECMs, but not via known major endocytic pathways, and suggest that such translocation may take place by diffusion of PNPs through the lipid bilayer of cell plasma membranes.

Observations of the distribution of GM3 in membrane microdomains by atomic force microscopy

Glycosphingolipids (GSLs) in membrane microdomains participate in important biological functions. In the present paper, we propose a novel model of the distribution of GSLs in membrane microdomains composed of sphingomyelin (SM) and cholesterol. We investigated the distribution of the ganglioside GM3 in a lipid membrane reconstituted with lipid extract from mouse B16 melanoma cells using an atomic force microscope (AFM). The surface topography of the reconstituted lipid bilayer showed three areas of different heights. The highest area was confirmed to be a GM3 domain by labeling with wheat germ agglutinin. To identify the lipids which are contributed to make the topography, the topographies of the artificial lipid bilayers composed of GM3, SM, 1-palmitoyl-2-oleoyl-phosphatidylcholine, and cholesterol were investigated. AFM images of the artificial lipid bilayers showed that the GM3 domain surrounded by a SM-containing phase only occurred, and its formation was found to depend on the cholesterol content.

Refolding and purification of the human secreted group IID phospholipase A 2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A 2 (sPLA 2s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A 2 (hsPLA 2-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA 2-IID in Escherichia coli, the DNA-coding sequence for hsPLA 2-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA 2-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A 2. The refolded recombinant hsPLA 2-IID demonstrated Ca 2+-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA 2-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.

HIV-1 Nef Perturbs Artificial Membranes: Investigation of the Contribution of the Myristoyl Anchor

Nef, an accessory protein from human immunodeficiency virus type 1, is critical for optimal viral replication and pathogenesis. Here, we analyzed the influence of full-length myristoylated and nonmyristoylated Nef on artificial lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By means of cosedimentation assays, we found that neither nonmyristoylated nor myristoylated Nef stably binds to POPC unilamellar vesicles. Time-resolved ellipsometry rather indicates that the proteins perturb the assembly of POPC planar bilayers. This observation was corroborated by fluorescence and scanning force microscopy, suggesting that membrane disordering occurs upon interaction of full-length myristoylated and nonmyristoylated Nef with planar POPC membranes immobilized on SiO2 surfaces resulting in loss of material from the surface. The membrane perturbations were further investigated by vesicle release experiments, demonstrating that the disordering results in defects through which the fluorophor carboxyfluorescein can pass. From these results, we conclude that Nef is capable of disordering and perturbing lipid membranes and that the myristoyl group is not the decisive determinant for the action of the protein on lipid membranes.

Amyloid-beta-induced ion flux in artificial lipid bilayers and neuronal cells: resolving a controversy.

Understanding the pathogenicity of amyloid-beta (Abeta) peptides constitutes a major goal in research on Alzheimer's disease (AD). One hypothesis entails that Abeta peptides induce uncontrolled, neurotoxic ion flux through cellular membranes. The exact biophysical mechanism of this ion flux is, however, a subject of an ongoing controversy which has attenuated progress toward understanding the importance of Abeta-induced ion flux in AD. The work presented here addresses two prevalent controversies regarding the nature of transmembrane ion flux induced by Alphabeta peptides. First, the results clarify that Alphabeta can induce stepwise ion flux across planar lipid bilayers as opposed to a gradual increase in transmembrane current; they show that the previously reported gradual thinning of membranes with concomitant increase in transmembrane current arises from residues of the solvent hexafluoroisopropanol, which is commonly used for the preparation of amyloid samples. Second, the results provide additional evidence suggesting that Abeta peptides can induce ion channel-like ion flux in cellular membranes that is independent from the postulated ability of Alphabeta to modulate intrinsic cellular ion channels or transporter proteins.

The M34A mutant of Connexin26 reveals active conductance states in pore-suspending membranes.

Connexin26 (Cx26) is a member of the connexin family, the building blocks for gap junction intercellular channels. These dodecameric assemblies are involved in gap junction-mediated cell-cell communication allowing the passage of ions and small molecules between two neighboring cells. Mutations in Cx26 lead to the disruption of gap junction-mediated intercellular communication with consequences such as hearing loss and skin disorders. We show here that a mutant of Cx26, M34A, forms an active hemichannel in lipid bilayer experiments. A comparison with the Cx26 wild-type is presented. Two different techniques using micro/nano-structured substrates for the formation of pore-suspending lipid membranes are used. We reconstituted the Cx26 wild-type and Cx26M34A into artificial lipid bilayers and observed single channel activity for each technique, with conductance levels of around 35, 70 and 165 pS for the wild-type. The conductance levels of Cx26M34A were found at around 45 and 70 pS.