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.     

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.     

Bilayer lipid membranes supported on Teflon filters: a functional environment for ion channels

Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100-300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter=1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8±0.2 nF and membrane resistances of 25.9±4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1-80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1-10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.     

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.     

Atomic force microscope image contrast mechanisms on supported lipid bilayers

This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.     

Spontaneous formation of two-dimensional and three-dimensional cholesterol crystals in single hydrated lipid bilayers

Grazing incidence x-ray diffraction measurements were performed on single hydrated bilayers and monolayers of Ceramide/Cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocyholine at varying concentrations. There are substantial differences in the phase and structure behavior of the crystalline domains formed within the bilayers relative to the corresponding monolayers, due to interactions between the opposing lipid leaflets. Depending on the lipid composition, these interactions lead to phase separation and formation of cholesterol crystals. The cholesterol and ceramide/cholesterol mixed phases were further characterized at 37°C by immunolabeling with specific antibodies recognizing ordered molecular arrays of cholesterol. Previous studies have shown that cholesterol may nucleate in artificial membranes to form thick two-dimensional bilayer crystals. The study herein demonstrates further growth of cholesterol into three-dimensional crystals. We believe that these results may provide further insight into the formation of cholesterol crystals in early stages of atherosclerosis inflammation.     

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.     

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.     

Molecular determinants for the line tension of coexisting liquid phases in monolayers

The line tension (λ) in biphasic membranes has been determined in monolayers and bilayers using a variety of techniques. In this work we present a novel approach to the determination of λ in monolayers with liquid/liquid phase coexistence, overcoming several of the drawbacks of current techniques. Using our method, we determined the line tension of liquid/liquid phases in binary mixtures of different lipids and a molecule similar to cholesterol but less oxidizable. We analyzed the effect of the hydrocarbon chain length and the polar head-group of the non-sterol lipid and found the latter to exert much more influence than the former. The presence of PE led to high λ values, PG to low values and PS and PC to intermediate values. The line tension showed a strong correlation with the critical packing parameter of the phospholipid. The spontaneous curvature displayed by the phases constituted by a particular lipid appears to be an important parameter for determining the line tension in mixed films.     

Miscibility of ternary mixtures of phospholipids and cholesterol in monolayers, and application to bilayer systems

We investigate miscibility transitions of two different ternary lipid mixtures, DOPC/DPPC/Chol and POPC/PSM/Chol. In vesicles, both of these mixtures of an unsaturated lipid, a saturated lipid, and cholesterol form micron-scale domains of immiscible liquid phases for only a limited range of compositions. In contrast, in monolayers, both of these mixtures produce two distinct regions of immiscible liquid phases that span all compositions studied, the alpha-region at low cholesterol and the beta-region at high cholesterol. In other words, we find only limited overlap in miscibility phase behavior of monolayers and bilayers for the lipids studied. For vesicles at 25 degrees C, the miscibility phase boundary spans portions of both the monolayer alpha-region and beta-region. Within the monolayer beta-region, domains persist to high pressures, yet within the alpha-region, miscibility phase transition pressures always fall below 15 mN/m, far below the bilayer equivalent pressure of 32 mN/m. Approximately equivalent phase behavior is observed for monolayers of DOPC/DPPC/Chol and for monolayers of POPC/PSM/Chol. As expected, pressure-area isotherms of our ternary lipid mixtures yield smaller molecular area and compressibility for monolayers containing more saturated acyl chains and cholesterol. All monolayer experiments were conducted under argon. We show that exposure of unsaturated lipids to air causes monolayer surface pressures to decrease rapidly and miscibility transition pressures to increase rapidly.     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

Recombinant human voltage-gated skeletal muscle sodium channels are pharmacologically functional in planar lipid bilayers

Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for ion channel research, high throughput screening for new drugs, and toxin detection. Protein suitable for these applications must be produced in a functionally active form. We report the successful use of ion metal affinity chromatography (IMAC) to purify C-terminal polyhistidine tagged human skeletal muscle voltage-gated sodium (hSkM1-HT) channels from Sf9 insect cells; hSkM1 channels were pharmacologically functional when reconstituted into liposomes and incorporated into planar bilayer lipid membranes. hSkM1-HT single channel currents activated by veratridine had a conductance of 21 pS and those activated by brevetoxin, 16 pS. Channel activity was inhibited by tetrodotoxin and saxitoxin. This protein is suitable for the development of biosensor and high throughput screening technologies.     

Reconstitution of the complement channel into lipid vesicles and planar bilayers starting from the fluid phase complex

Proteolysis of the fluid phase complement complex SC5b-9 transforms it into an arnphiphilic molecule which resembles the membrane attack complex of complement and reconstitutes into lipid vesicles. Complement-containing vesicles prepared in this way can be made to fuse with planar lipid bilayers transferring their protein content to the host membrane. Massive conductance increases can thus be observed, which are due to the insertion of a large number of ionic channels into the membrane. Using low concentrations of vesicles, single channels can be studied.     

Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers.

Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they were blocked by internally applied "ball" inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.     

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.     

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.     

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.     

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.