Discrete membrane arrays

This review describes various methods for the attachment of phospholipid bilayers to solid supports. The simplest approach involves vesicle unrolling onto a surface that has been previously modified with a continuous self-assembled monolayer (SAM). The choice of a suitable SAM can lead to the formation of attached bilayers that have the desired biomimetic properties and are suitable for studying transmembrane proteins. However, there are intrinsic problems associated with this approach if one is interested in studying ion transport phenomena. In particular, the relatively low resistance values found for such bilayers do not permit studies of single ion channels. For such studies to be carried out the background leakage through the lipid film must be greatly reduced. In an attempt to reduce the problems of leakage we have formed patterned SAMs in which a blocking, hydrophobic, layer covers 90% of the electrode surface. The remaining portion of the surface, which is hydrophilic, supports the formation of a bilayer. This approach has led to an improvement in the quality of the bilayers formed but has still not provided bilayers with sufficiently high specific resistances to study single ion channels. Finally, we describe new approaches based on the formation of bilayers suspended over small apertures. These ‘suspended’ bilayers are similar in structure to those used in black lipid membrane experiments and give rise to highly blocking bilayer membranes. Unfortunately, this approach requires the use of solvents to create the suspended bilayer and they are relatively fragile.     

Lipid bilayer arrays: Cyclically formed and measured

Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, and can potentially be applied in DNA sequencing. Droplet bilayers have significant technological potential for enabling many of these applications due to their compatibility with automation and array platforms. To further develop this potential, we have simplified the formation and electrical measurement of droplet bilayers using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and measurement over a 32‐element array with ～80% yield and no operator input following fluid addition. Cycling these arrays resulted in the formation and measurement of 96 out of 120 possible bilayers in 80 minutes, a sustainable rate that could significantly increase with automation and greater parallelization. This turn‐key, high‐yield approach to making artificial lipid bilayers requires no training, making the capability of creating and measuring lipid bilayers and ion channels accessible to a much wider audience. In addition, this approach is low‐cost, parallelizable, and automatable, allowing high‐throughput studies of ion channels and pore proteins in lipid bilayers for sensing or screening applications.     

Ion channel and toxin measurement using a high throughput lipid membrane platform

Measurements of ion channels are important for scientific, sensing and pharmaceutical applications. Reconstitution of ion channels into lipid vesicles and planar lipid bilayers for measurement at the single molecule level is a laborious and slow process incompatible with the high throughput methods and equipment used for sensing and drug discovery. A recently published method of lipid bilayer formation mechanically combines lipid monolayers self-assembled at the interfaces of aqueous and apolar phases. We have expanded on this method by vertically orienting these phases and using gravity as the driving force to combine the monolayers. As this method only requires fluid dispensation, it is trivially integrated with high throughput automated liquid-handling robotics. In a proof-of-concept demonstration, we created over 2200 lipid bilayers in 3h. We show single molecule measurements of technologically and physiologically relevant ion channels incorporated into lipid bilayers formed with this method.     

Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip

Painted bilayers containing reconstituted ion channels serve as a well defined model system for electrophysiological investigations of channel structure and function. Horizontally oriented bilayers with easy solution access to both sides were obtained by painting a phospholipid:decane mixture across a cylindrical pore etched into a 200-microm thick silicon wafer. Silanization of the SiO(2) layer produced a hydrophobic surface that promoted the adhesion of the lipid mixture. Standard lithographic techniques and anisotropic deep-reactive ion etching were used to create pores with diameters from 50 to 200 microm. The cylindrical structure of the pore in the partition and the surface treatment resulted in stable bilayers. These were used to reconstitute Maxi K channels in the 100- and 200-microm diameter pores. The electrophysiological characteristics of bilayers suspended in microchips were comparable with that of other bilayer preparations. The horizontal orientation and good voltage clamping properties make the microchip bilayer method an excellent system to study the electrical properties of reconstituted membrane proteins simultaneously with optical probes.     

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.     

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes

The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 μs and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.     

Properties of ionic channels formed by the antibiotic syringomycin E in lipid bilayers: dependence on the electrolyte concentration in the bathing solution.

Using the planar lipid bilayer technique, organization of ionic channels formed by the lipodepsipeptide antibiotic syringomycin E applied to one (cis) side of a lipid bilayer was studied. Low concentrations of NaCl (0.01-0.1 M) induced the opening and closing of two types of channels - "small" and "large". The large channels had single channel conductances approximately six times greater than those of the small channels. An increase in the NaCl concentration (0.6-1.0 M) decreased almost completely the chance to reveal the large channels. Although the syringomycin channels exhibited the anion selectivity within the entire range of NaCl concentrations in the bathing solutions (from 0.001 to 1.0 M) whereas the concentration gradients across the bilayers were 2 and 4, the transfer numbers for Cl-decreased with an increase in the mean NaCl concentration (from 0.83 for 0.005 M to 0.70 for 0.5 M). Moreover, at each mean value of NaCl concentration, all conductance levels had the same ion selectivity (identical reversal potential). These results suggest that at low NaCl concentrations the large channels are clusters of small channels which synchronously open and close, while at high electrolyte concentrations the screening of the charged groups responsible for channel interactions prevents the cluster formation. A new theoretical approach for the estimation of the channel radius and the number of elementary charges located at its inner surface (based on the experimental curve of the dependence of transfer number on the NaCl concentration) was developed. Based on this theoretical approach, the channel radius equal to 1 nm and one elementary charge located at its inner surface were obtained.     

Pure lipid vesicles can induce channel-like conductances in planar bilayers

Summary Typical channel-like current fluctuations were observed in planar lipid bilayers following brief exposure to large concentrations of lipid vesiclesdevoid of protein. Vesicles, formed by sonication of pure lipids suspended in 150mm salt solutions, were ejected 0.5 mm from a planar bilayer with a pipette. Over the next several minutes the bilayer conductance changed in ways usually considered to be indicative of reconstituted protein channels including step conductance changes (both up and down), flickering, ion selectivity, and inactivation. This observation demonstrates the need for caution in interpreting conductance changes which occur following ejection of channel-containing vesicles near a membrane.     

Conformational and orientation studies of artificial ion channels incorporated into lipid bilayers

The conformational and orientation studies in lipid bilayers of 21 amino acid peptides bearing six crown ethers are reported. The compounds were designed to form artificial ion channels by stacking the crown rings, and were shown to be functional in bilayer membranes. We used Fourier transform infrared spectroscopy and CD spectropolarimetry to study the conformation of the peptides in solution and in lipid bilayers. These studies revealed that hexacrown peptides retain their alpha-helical conformation when incorporated in a lipid bilayer environment. Attenuated total reflectance spectroscopy was used to investigate the orientation of the peptides in a lipid bilayer. Results demonstrated that the peptides are not oriented at a fixed angle in membrane, but rather are in incorporation equilibrium between an active state parallel to the lipid chain and an inactive state adsorbed at the surface of the bilayer. From these results, we propose a model for the channel activity and the gating mechanism of these hexacrown peptides in bilayer membranes.     

Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat

Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn(2+). DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn(2+)-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.     

Homogeneous hybrid droplet interface bilayers assembled from binary mixtures of DPhPC phospholipids and PB-b-PEO diblock copolymers

Hybrid membranes built from phospholipids and amphiphilic block copolymers seek to capitalize on the benefits of both constituents for constructing biomimetic interfaces with improved performance. However, hybrid membranes have not been formed or studied using the droplet interface bilayer (DIB) method, an approach that offers advantages for revealing nanoscale changes in membrane structure and mechanics and offers a path toward assembling higher-order tissues. We report on hybrid droplet interface bilayers (hDIBs) formed in hexadecane from binary mixtures of synthetic diphytanoyl phosphatidylcholine (DPhPC) lipids and low molecular weight 1,2 polybutadiene-b-polyethylene oxide (PBPEO) amphiphilic block copolymers and use electrophysiology measurements and imaging to assess the effects of PBPEO in the membrane. This work reveals that hDIBs containing up to 15 mol% PBPEO plus DPhPC are homogeneously mixtures of lipids and polymers, remain highly resistive to ion transport, and are stable—including under applied voltage. Moreover, they exhibit hydrophobic thicknesses similar to DPhPC-only bilayers, but also have significantly lower values of membrane tension. These characteristics coincide with reduced energy of adhesion between droplets and the formation of alamethicin ion channels at significantly lower threshold voltages, demonstrating that even moderate amounts of amphiphilic block copolymers in a lipid bilayer provide a route for tuning the physical properties of a biomimetic membrane.     

beta-Sheet structured beta-amyloid(1-40) perturbs phosphatidylcholine model membranes

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Abeta40 assemblies.     

Calcium-binding to non-ionic lipids: crystal structure of a calcium chloride complex of geraniol.

X-ray diffraction data were used to determine the crystal structure of a calcium chloride complex of geraniol. The geraniol molecules assume a bilayer arrangement, with channels of calcium and chloride ions separating the bilayers. Each calcium ion is coordinated to the hydroxyl groups of two symmetry-related geraniol molecules and to four chloride ions. Our results demonstrate that hydrophobic interactions within a lipid bilayer can lead to an arrangement of hydroxyl groups suitable for binding calcium ions. Similar interactions may be involved in the calcium-binding sites on membrane surfaces.     

Automatable production of shippable bilayer chips by pin tool deposition for an ion channel measurement platform

As a step towards an automated and operator-free ion channel measurement platform we have previously demonstrated a solution formulation for artificial lipid bilayers that enabled the indefinite storage and shipping of frozen bilayer precursors. In this work, the solutions were deposited by hand. Here, we have adapted pin tools to deposit the bilayer precursor solutions onto multi-element arrays, a popular method for microarray solution deposition. The pin tools have enabled the deposited volume to be applied highly repeatably and controllably, resulting in reduction of bilayer formation times to <1 h. The pin tools are also compatible with computerized motion control platforms, enabling automated and high throughput production. We discuss these results and the prospects of this technology to produce high density bilayer arrays for high throughput measurement of ion channels incorporated into artificial bilayers.     

A tethered bilayer sensor containing alamethicin channels and its detection of amiloride based inhibitors

Alamethicin, a small transmembrane peptide, inserts into a tethered bilayer membrane (tBLM) to form ion channels, which we have investigated using electrical impedance spectroscopy. The number of channels formed is dependent on the incubation time, concentration of the alamethicin and the application of DC voltage. The properties of the ion channels when formed in tethered bilayers are similar to those for such channels assembled into black lipid membranes (BLMs). Furthermore, amiloride and certain analogs can inhibit the channel pores, formed in the tBLMs. The potency and concentration of the inhibitors can be determined by measuring the change of impedance. Our work illustrates the possibility of using a synthetic tBLM for the study of small peptide voltage dependent ion channels. A potential application of such a device is as a screening tool in drug discovery processes.     

Kinetics of opening and closure of syringomycin E channels formed in lipid bilayers

A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels.