Bilayer-gel membranes. Formation and some properties

We developed a new procedure which induces multifunctional reagents to crosslink at one interface between a black bilayer and the adjacent water phase. This procedure yields ‘bilayer-gel’ membranes, i.e. membranes consisting of a bilayer and a polymer layer. The bilayer-gel membrane may tentatively be considered to be a new membrane system, because the formation of the polymer layer changes some bilayer properties. We studied bilayer-gel membranes composed of a bilayer of oxidized cholesterol and of a polymer layer of poly-l-lysine crosslinked by glutardialdehyde. Compared to unmodified bilayers, this membrane system has an electrical conductance of the same magnitude, the same electrical capacity and similar shapes of current-voltage dependences. However, this system is asymmetrical and differs in ion selectivity and increased stability from an unmodified bilayer.     

Bilayer-gel membranes. Formation and some properties.

We developed a new procedure which induces multifunctional reagents to crosslink at one interface between a black bilayer and the adjacent water phase. This procedure yields "bilayer-gel" membranes, i.e. membranes consisting of a bilayer and a polymer layer. The bilayer-gel membrane may tentatively be considered to be a new membrane system, because the formation of the polymer layer changes some bilayer properties. We studied bilayer-gel membranes composed of a bilayer of oxidized cholesterol and of a polymer layer of poly-L-lysine crosslinked by glutardialdehyde. Compared to unmodified bilayers, this membrane system has an electrical conductance of the same magnitude, the same electrical capacity and similar shapes of current-voltage dependences. However, this system is asymmetrical and differs in ion selectivity and increased stability from an unmodified bilayer.     

A molecular toolkit for highly insulating tethered bilayer lipid membranes on various substrates

Tethered bilayer lipid membranes (tBLMs) are promising model architectures that mimic the structure and function of natural biomembranes. They provide a fluid, stable, and electrically sealing platform for the study of membrane related processes, specifically, the function of incorporated membrane proteins. This paper presents a generic approach toward the synthesis of functional tBLMs adapted for application to various surfaces. The central element of a tethered membrane consists of a lipid bilayer. Its proximal layer is covalently attached via a spacer unit to a solid support, either gold or silicon oxide. The membranes are characterized optically by using surface plasmon resonance spectroscopy (SPR) or ellipsometry and electrically by using electrochemical impedance spectroscopy (EIS). The bilayer membranes obtained show high electrical barrier properties and can be used to incorporate and study small membrane proteins in a functional form.     

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.     

Signal transduction across alamethicin ion channels in the presence of noise.

We have studied voltage-dependent ion channels of alamethicin reconstituted into an artificial planar lipid bilayer membrane from the point of view of electric signal transduction. Signal transduction properties of these channels are highly sensitive to the external electric noise. Specifically, addition of bandwidth-restricted "white" noise of 10-20 mV (r.m.s.) to a small sine wave input signal increases the output signal by approximately 20-40 dB conserving, and even slightly increasing, the signal-to-noise ratio at the system output. We have developed a small-signal adiabatic theory of stochastic resonance for a threshold-free system of voltage-dependent ion channels. This theory describes our main experimental findings giving good qualitative understanding of the underlying mechanism. It predicts the right value of the output signal-to-noise ratio and provides a reliable estimate for the noise intensity corresponding to its maximum. Our results suggest that the alamethicin channel in a lipid bilayer is a good model system for studies of mechanisms of primary electrical signal processing in biology showing an important feature of signal transduction improvement by a fluctuating environment.     

Molecular dynamics simulations of salicylate effects on the micro- and mesoscopic properties of a dipalmitoylphosphatidylcholine bilayer

Salicylate, an amphiphilic molecule and a popular member of the nonsteroidal anti-inflammatory drug family, is known to affect hearing through reduction of the electromechanical coupling in the outer hair cells of the ear. This reduction of electromotility by salicylate has been widely studied, but the molecular mechanism of the phenomenon is still unknown. In this study, we investigated one aspect of salicylate's action, namely the perturbation of electrical and mechanical membrane properties by salicylate in the absence of cytoskeletal or membrane-bound motor proteins such as prestin. In particular, we simulated the interaction of salicylate with a dipalmitoylphosphatidylcholine (DPPC) bilayer via atomically detailed molecular dynamics simulations to observe the effect of salicylate on the microscopic and mesoscopic properties of the bilayer. The results demonstrate that salicylate interacts with the bilayer by associating at the water-DPPC interface in a nearly perpendicular orientation and penetrating more deeply into the bilayer than either sodium or chloride. This association has several affects on the membrane properties. First, binding of salicylate to the membrane displaces chloride from the bilayer-water interface. Second, salicylate influences the electrostatic potential and dielectric properties of the bilayer, with significant changes at the water-lipid bilayer interface. Third, salicylate association results in structural changes, including decreased headgroup area per lipid and increased lipid tail order. However, salicylate does not significantly alter the mechanical properties of the DPPC bilayer; bulk compressibility, area compressibility, and bending modulus were only perturbed by small, statistically insignificant amounts by the presence of salicylate. The observations from these simulations are in qualitative agreement with experimental data and support the conclusion that salicylate influences the electrical but not the mechanical properties of DPPC membranes.     

The effect of temperature on capacitance changes in an oscillating model membrane

The electrical properties of model membranes are altered during stretching or pressure pulses. We have used a mechanico-electric transduction model to interpret the temperature dependence of capacitance changes produced in oxidized cholesterol membranes during mechanical oscillation. The relative contribution of the torus and bilayer portions of the membrane to the capacitance change is identified. The difference in elasticity between the bilayer and torus decreases rapidly with decreasing temperature and ultimately the torus becomes as solid as the bilayer portion of the model membrane.     

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.     

Amphiphiles capsaicin and triton X-100 regulate the chemotherapy drug colchicine’s membrane adsorption and ion pore formation potency

Chemotherapy drugs (CDs), e.g. colchicine derivative thiocolchicoside (TCC) and taxol, have been found to physically bind with lipid bilayer membrane and induce ion pores. Amphiphiles capsaicin (Cpsn) and triton X-100 (TX100) are known to regulate lipid bilayer physical properties by altering bilayer elasticity and lipid monolayer curvature. Both CDs and amphiphiles are predicted to physically accommodate alongside lipids in membrane to exert their membrane effects. The effects of their binary accommodation in the lipid membrane are yet to be known. Firstly, we have performed experimental studies to inspect whether membrane adsorption of CDs (colchicine or TCC) gets regulated due to any membrane effects of Cpsn or TX100. We find that the aqueous phase presence of these amphiphiles, known to reduce the membrane stiffness, works towards enhancing the membrane adsorption of CDs. Our recently patented technology ‘direct detection method’ helps address the membrane adsorption mechanisms. Secondly, in electrophysiology records, we measured the amphiphile effects on the potency of ion channel induction due to CDs. We find that amphiphiles increase the CD induced channel induction potency. Specifically, the membrane conductance, apparently due to the ion channel induction by the TCC, increases substantially due to the Cpsn or TX100 induced alterations of the bilayer physical properties. Thus we may conclude that the binary presence of CDs and amphiphiles in lipid membrane may influence considerably in CD’s membrane adsorption, as well as the membrane effects, such as ion pore formation.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

The OpenPicoAmp: An Open-Source Planar Lipid Bilayer Amplifier for Hands-On Learning of Neuroscience

Understanding the electrical biophysical properties of the cell membrane can be difficult for neuroscience students as it relies solely on lectures of theoretical models without practical hands on experiments. To address this issue, we developed an open-source lipid bilayer amplifier, the OpenPicoAmp, which is appropriate for use in introductory courses in biophysics or neurosciences at the undergraduate level, dealing with the electrical properties of the cell membrane. The amplifier is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. In addition, we propose a specific design for experimental chambers allowing the insertion of a commercially available polytetrafluoroethylene film. We provide a complete documentation allowing to build the amplifier and the experimental chamber. The students hand-out giving step-by step instructions to perform a recording is also included. Our experimental setup can be used in basic experiments in which students monitor the bilayer formation by capacitance measurement and record unitary currents produced by ionic channels like gramicidin A dimers. Used in combination with a low-cost data acquisition board this system provides a complete solution for hands-on lessons, therefore improving the effectiveness in teaching basic neurosciences or biophysics.     

The effect of hexadecaprenol on molecular organisation and transport properties of model membranes

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.     

Insulating tethered bilayer lipid membranes to study membrane proteins

Tethered bilayer lipid membranes are stable and promising model systems that mimic several properties of biological membranes. They provide an electrically insulating platform for the incorporation and study of functional membrane proteins, especially ion channels. Covalently linked to a solid support, they also offer enhanced stability compared with other model architectures. If the support can be used as an electrode, electrical characterisation of the system is possible and biosensing applications can be envisioned.Here, we will review some tethered bilayer structures developed in the past and show some examples of functional protein incorporation, both on oxide and gold substrates.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

Voltage-induced gating of the mechanosensitive MscL ion channel reconstituted in a tethered lipid bilayer membrane

The mechanosensitive (MS) ion channel is gated by changes in bilayer deformation. It is functional without the presence of any other proteins and gating of the channel has been successfully achieved using conventional patch clamping techniques where a voltage has been applied together with a pressure over the membrane. Here, we have for the first time analyzed the large conducting (MscL) channel in a supported membrane using only an external electrical field. This was made possible using a newly developed technique utilizing a tethered lipid bilayer membrane (tBLM), which is part of an engineered microelectronic array chip. Single ion channel activity characteristic for MscL was obtained, albeit with lower conductivity. The ion channel was gated using solely a transmembrane potential of 300 mV. Computations demonstrate that this amount of membrane potential induces a membrane tension of 12 dyn/cm, equivalent to that calculated to gate the channel in patch clamp from pressure-induced stretching of the bilayer. These results strengthen the supposition that the MscL ion channel gates in response to stress in the lipid membrane rather than pressure across it. Furthermore, these findings illustrate the possibility of using the MscL as a release valve for engineered membrane devices; one step closer to mimicking the true function of the living cell.     

Connexins and their environment: effects of lipids composition on ion channels

Intercellular communication is mediated through paired connexons that form an aqueous pore between two adjacent cells. These membrane proteins reside in the plasma membrane of their respective cells and their activity is modulated by the composition of the lipid bilayer. The effects of the bilayer on connexon structure and function may be direct or indirect, and may arise from specific binding events or the physicochemical properties of the bilayer. While the effects of the bilayer and its constituent lipids on gap junction activity have been described in the literature, the underlying mechanisms of the interaction of connexin with its lipidic microenvironment are not as well characterized. Given that the information regarding connexons is limited, in this review, the specific roles of lipids and the properties of the bilayer on membrane protein structure and function are described for other ion channels as well as for connexons.     

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.     

Connexins and their environment: effects of lipids composition on ion channels

Intercellular communication is mediated through paired connexons that form an aqueous pore between two adjacent cells. These membrane proteins reside in the plasma membrane of their respective cells and their activity is modulated by the composition of the lipid bilayer. The effects of the bilayer on connexon structure and function may be direct or indirect, and may arise from specific binding events or the physicochemical properties of the bilayer. While the effects of the bilayer and its constituent lipids on gap junction activity have been described in the literature, the underlying mechanisms of the interaction of connexin with its lipidic microenvironment are not as well characterized. Given that the information regarding connexons is limited, in this review, the specific roles of lipids and the properties of the bilayer on membrane protein structure and function are described for other ion channels as well as for connexons.     

Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers

Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.     

Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes

The action of the 107 kDa hemolysin from Escherichia coli on planar lipid membranes was investigated. We report that a single toxin molecule can form a cation-selective, ion-permeable channel of large conductance in a planar phospholipid bilayer membrane. The conductance of the pore is proportional to that of the bulk solution, indicating that the channel is filled with water. A pore diameter of about 2 nm can be evaluated. The pore formation mechanism is voltage-dependent and essentially resembles that of pore-forming colicins; this implies that opening of the channel is dependent on transfer of an electrical charge through the membrane. We propose that the physiological effects of E. coli hemolysin result from its ability to form ion channels in the membrane of attacked cells, and show that there is quantitative agreement between the effects of this toxin on model membranes and its hemolytic properties.