Planar Bilayer System Research Articles

Nanoparticles of Push-Pull Triphenylamine-Based Molecules for Light-Controlled Stimulation of Neuronal Activity.

Organic semiconductor materials with a unique set of properties are very attractive for interfacing biological objects and can be used for noninvasive therapy or detection of biological signals. Here, we describe the synthesis and investigation of a novel series of organic push-pull conjugated molecules with the star-shaped architecture, consisting of triphenylamine as a branching electron donor core linked through the thiophene π-spacer to electron-withdrawing alkyl-dicyanovinyl groups. The molecules could form stable aqueous dispersions of nanoparticles (NPs) without the addition of any surfactants or amphiphilic polymer matrixes with the average size distribution varying from 40 to 120 nm and absorption spectra very similar to those of human eye retina pigments such as rods and green cones. Variation of the terminal alkyl chain length of the molecules forming NPs from 1 to 12 carbon atoms was found to be an efficient tool to modulate their lipophilic and biological properties. Possibilities of using the NPs as light nanoactuators in biological systems or as artificial pigments for therapy of degenerative retinal diseases were studied both on the model planar bilayer lipid membranes and on the rat cortical neurons. In the planar bilayer system, the photodynamic activity of these NPs led to photoinactivation of ion channels formed by pentadecapeptide gramicidin A. Treatment of rat cortical neurons with the NPs caused depolarization of cell membranes upon light irradiation, which could also be due to the photodynamic activity of the NPs. The results of the work gave more insight into the mechanisms of light-controlled stimulation of neuronal activity and for the first time showed that fine-tuning of the lipophilic affinity of NPs based on organic conjugated molecules is of high importance for creating a bioelectronic interface for biomedical applications.

Understanding Transport of Antibiotic Molecules Across Outer Membrane Channels in Their Native Environment Using Outer Membrane Vesicle Fusion as a Method

Antimicrobial resistance is one of the most severe health concerns worldwide. This resistance is often caused by a reduced uptake through outer membrane channels. A method to characterize flux is to extract and reconstitute outer membrane channels into a planar bilayer system. Here in this study, we show a robust approach by directly fusing outer membrane vesicles with over expressed OmpF from Escherichia coli into a planar bilayer system, which allows us to characterize outer membrane channels in their native environment. Typically single OMV fusion are observed by single channel activity (1-5 channel at a time). After successful fusion with the bilayer, the LPS originated from the OMV's introduces an asymmetry on the bilayer, LPS being present dominantly on the side of OMV addition. Compared to conventional methods, the channels present in the OMVs containing LPS have a wider range of conductance. Further, we characterize the permeation of Norfloxacin, Ciprofloxacin, Enrofloxacin and Kanamycin Sulfate through these channels. Addition of these molecules on the LPS side yields lower on-rates compared with the conventional, LPS free reconstitution. We conclude that using outer membrane vesicle fusion with a planar bilayer is a fast and robust approach for studying membrane channels in their native environment.

Effect of fullerene on the dispersibility of nanostructured lipid particles and encapsulation in sterically stabilized emulsions

We report on the effect of fullerenes (C60) on the stability of nanostructured lipid emulsions. These (oil-in-water) emulsions are essentially aqueous dispersions of lipid particles exhibiting self-assembled nanostructures at their cores. The majority of previous studies on fullerenes were focused on planar and spherical lipid bilayer systems including pure lipids and liposomes. In this work, fullerenes were interacted with a lipid that forms nanostructured dispersions of non-lamellar self-assemblies. A range of parameters including the composition of emulsions and sonication parameters were examined to determine the influence of fullerenes on in-situ and pre-stabilized lipid emulsions. We found that fullerenes mutually stabilize very low concentrations of lipid molecules, while other concentration emulsions struggle to stay stable or even to form at first instance; we provide hypotheses to support these observations. Interestingly though, we were able to encapsulate varying amounts of fullerenes in sterically stabilized emulsions. This step has a significant positive impact, as we could effectively control an inherent aggregation tendency of fullerenes in aqueous environments. These novel hybrid nanomaterials may open a range of avenues for biotechnological and biomedical applications exploiting properties of both lipid and fullerene nanostructures.

Robust reagent addition and perfusion strategies for droplet-interface bilayers

We have designed two novel devices which extend the applications for the droplet-interface bilayer (DIB) as a model membrane system. The add-chip allows successive reagent additions to one side of the lipid bilayer during an experiment while maintaining a simple setup with much lower volumes than in planar bilayer systems. The flow-chip is capable of multiple complete solution perfusions concurrently with electrophysiology measurements. Both devices preserve all of the key advantages that DIBs have relative to planar membranes, including low volume, leaflet asymmetry and the ability to separate the monolayers prior to further analysis of a droplet's contents. As a demonstration, we use these devices to monitor and quantitate molecular transport across DIBs.

Curvature Driven Membrane Organisation Revealed by Single Molecule Imaging in Droplet Interface Bilayers

Membrane curvature plays a crucial role in lipid and protein sorting within the cell membrane [1] giving rise to spontaneous self-organisation and localisation of complex biological macromolecules. Such spatial organisation in response to, and in generating, membrane curvature contributes to many important biological processes such as neurotransmission, trafficking and cell division [2]. Despite this importance, the majority of in-vitro studies are performed in planar or pseudo-planar (GUV) bilayer systems. As such, there is a need for novel strategies to more closely mimic the curvature of cellular membranes, whilst allowing for correlated measurements of protein function.Here, we create Droplet Interface Bilayers (DIBs) [3] on an underlying curved substrate, resulting in the morphology of the underlying substrate being reflected in the curvature of the bilayer. Total Internal Reflection Fluorescence (TIRF) imaging reveals the correlation of lipid domain formation at sites of intrinsic curvature and the preferential localisation and pore formation by antimicrobial peptides at regions of positive curvature. This technique provides a new method for dictating the curvature of artificial bilayers, enabling single molecule studies on the role of curvature in membrane organisation and function.1. Ramamurthi, K. S. et al. Science 2009 323, 1354.2. McMahon, H. T. et al. Nature 2005 438, 590.3. Thompson, J. R. et al. Nano Letters 2007 7, 3875.

On-Chip Stochastic Resonance of Ion Channel Systems With Variable Internal Noise

We induced stochastic resonance in planar lipid bilayer systems with alamethicin ion channels, and varied alamethicin concentration, membrane area, and applied voltage. We found that membrane-induced microphonic noise significantly affects the signature of stochastic resonance, and that this noise can be used to optimize ion channel-based biosensors.

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, and PLCγ15.In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3ζ, ZAP70, LAT, SLP-76, Itk, and PLCγ15. In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

Cooperativity of SNARE Complexes in Membrane Fusion: Mechanisms of SNARE Cluster-Mediated Docking and Fusion

SNARE proteins are the core of the cellular fusion machinery. Vesicle v-SNAREs engage target membrane t-SNAREs and form SNARE complexes thought to pull the membrane surfaces together and drive fusion. Evidence suggests that many SNARE complexes participate in single fusion events and in SNARE-reconstituted vesicle-supported bilayer fusion assays 5-11 complexes were required for fusion [Karatekin et al, PNAS, 2010]. However a mechanistic understanding of this requirement is lacking. We have developed a mathematical model of SNARE cluster-driven docking and fusion which explicitly accounts for interactions between the participating SNARE complexes and the intermembrane forces that must be overcome to trigger fusion. The model is analyzed by a combination of analytical methods and computer simulations. We find that the energetically-favored cluster configuration is a ring of completely assembled SNARE complexes which dock the vesicle to the target membrane. Simulations show that a minimally-sized cluster ring expands outward, driven principally by electrostatic and steric repulsions between the SNARE complexes. Ring expansion thereby pulls the curved vesicle surface toward the target membrane. Addition of new complexes to the cluster further expands the ring, thus reducing the membrane separation and elevating the pressure between the membranes. We find a cluster of ∼5-10 complexes docking a 50-nm vesicle exerts local pressures of tens of atmospheres, similar to the threshold pressures required for fusion measured in planar bilayer systems [Wong et al, Biophys J., 1999]. Thus, the SNARE cluster-generated pressure may be sufficient for fusion. This is consistent with the recently reported 5-11 complex requirement. In addition, our model makes the testable prediction that fusion of smaller (higher curvature) vesicles requires fewer SNAREs.

Multiphoton excitation fluorescence microscopy in planar membrane systems

The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

Simulations of the Morphological Evolution of Lipid Bilayer Membranes Using a Phase-Field Method

We present a continuum-level simulation method for modeling phase separation and morphological evolution of multicomponent lipid bilayer membranes. Our objective is to investigate how various physical parameters input into the model, such as spontaneous curvature, phase fraction, and interleaflet coupling strength, affect the dynamics and equilibrium morphological phases formed in two-phase lipid bilayer membrane systems. The model applies to membranes with planar and spherical background geometries, simulating a nearly planar portion of membrane or entire vesicle, respectively. The compositions and shape of the membrane are coupled through a modified Helfrich free energy. The planar model treats the composition of each leaflet, and thus includes a term coupling these compositions across the bilayer. The compositional evolution is modeled using a phase-field method and is described by a Cahn-Hilliard-type equation, while shape changes are described by relaxation dynamics. For nearly planar bilayer systems with each leaflet having the same phase fraction, we find that domains in both leaflets align to reduce the interaction energy, as expected. When the coupling effect is stronger, this alignment occurs more quickly and more precisely, showing that the coupling affects the dynamics. This leaflet coupling is found to heavily influence morphological evolution; in some cases the equilibrium morphological phase observed is very different from what was observed with our simpler monolayer model using similar conditions. We construct a phase diagram of equilibrium morphological phases in the composition space for a few values of the strength of the leaflet coupling. This model has been able to reproduce results found in lipid bilayer experiments probing interleaflet interactions, including the effect of domain registration across leaflets. For the vesicle model, we investigate how an ellipsoidal geometry imposed in the initial conditions affects the phase and morphological evolution.

Exciton Diffusion Length and Lifetime in Subphthalocyanine Films

Organic photovoltaic cells with chloro[subphthalocyaninato]boron(III) (SubPc) as donor in combination with buckminster fullerene (C60) as acceptor in a planar heterojunction configuration have shown high power conversion efficiencies. The parameter that determines the critical distance to the heterojunction over which light-generated excitons can be harvested is the exciton diffusion length. Here, we characterize this exciton diffusion length in SubPc thin films by photoluminescence (PL) quenching experiments in a planar bilayer system. As the layer thickness is much smaller than the optical penetration depth, we apply the transfer matrix method (TMM) to calculate the internal optical field inside the organic structures and use this to derive the intrinsic PL spectrum. An exciton diffusion length of 28 nm is determined, and good agreement is found with the diffusion lengths obtained from modeling the external quantum efficiency. In addition, time-resolved PL measurements are performed to determine the exc...

Nanoscale Increases in CD2-CD48-mediated Intermembrane Spacing Decrease Adhesion and Reorganize the Immunological Synapse

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity.

Magnetic phase diagrams for models of synthetic antiferromagnets

Phenomenological macrospin models have been studied to elucidate the field-driven reorientation transitions in antiferromagnetically coupled magnetic thin-film elements with uniaxial anisotropy. For planar bilayer systems, there are four topologically different phase diagrams depending on the ratio of anisotropy to exchange energy. Only for weak anisotropies, the usual phase diagram of antiferromagnets with a spin flop is realized. Analytic and numeric results are presented for a model of a synthetic antiferromagnet composed of four layers, where two ferromagnetically coupled bilayers are coupled antiferromagnetically. Applied fields may stabilize collinear ferrimagnetic and canted phases in these four-layer systems for certain ranges of the ratio between ferromagnetic exchange and antiferromagnetic exchange.

Controlled delivery of proteins into bilayer lipid membranes on chip.

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.

Gramicidin-based channel systems for the detection of protein–ligand interaction

To detect protein–ligand interaction a gramicidin-based sensor was developed. Biotin was tagged to the C-terminus of gramicidin (Gram-bio 1). The biotin-moiety, which faces the electrolyte, gave little effect on single-channel conductance. Streptavidin added to the electrolyte was detected by Gram-bio 1 through the monitoring channel current using the planar bilayer system. The suppression of macroscopic currents and the acceleration of their decaying time course were observed in a concentration dependent manner. In the single-channel level, however, no significant effect on the single-channel conductance and the open dwell time was observed upon addition of streptavidin. Therefore, streptavidin neither blocked the open channel nor changed the stability of the conducting dimer. Insertion of a linker between gramicidin and biotin did not change the streptavidin-sensitivity of the current reduction. We conclude that the binding of streptavidin to the Gram-bio 1 shifted the distribution of the complex from the membrane to the electrolyte and, thus, reduced the formation of conducting dimer of Gram-bio 1 in the membrane. Interaction of biotin with an anti-biotin antibody was also observed using this system, indicating that this system is applicable for the detection of protein–ligand interaction having a binding constant of ∼10 8–9 M −1 or more. Both the adamantane-tagged gramicidin for detection of β-cyclodextrin and the Strep Tag-II-tagged gramicidin for detection of streptavidin (binding constant: ∼10 5 M −1 or less) failed to respond. Thus, high-affinity ligands upon tagging to gramicidin render the gramicidin-based sensor able to execute as a real-time monitoring system for protein–ligand interaction.

How To Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy

Confocal fluorescence correlation spectroscopy (FCS) allows for the determination of lateral diffusion coefficients and surface densities in planar phospholipid systems. The determination of the vertical (z-) position of the laser focus relative to the phospholipid surface plane is of crucial importance for the accuracy of the confocal FCS experiment. In this work we determine for the first time this vertical (z-) position of the laser focus by a so-called “Z-scan”, which is based on the determination of diffusion times and particle numbers in 0.2 μm steps along the vertical (z-) axis. Experiments on supported phospholipid bilayers composed of dioleoylphosphatidylcholine (DOPC) and small amounts of Rhodamine Red-X 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Rhodamine Red-X DHPE) adsorbed onto atomically flat mica and borosilicate glass demonstrate that results obtained by the Z-scan approach are significantly more precise than those results obtained when the fluorescence intensity maximum is used as an indicator in the determination of the vertical (z-) position of the sample. In addition to this basic contribution for the investigation of planar bilayer systems by confocal FCS, the lateral diffusion coefficients of Rhodamine Red-X DHPE in supported phospholipid bilayers composed of DOPC and cholesterol as well as in DOPC or dipalmitoylphosphatidylcholine (DPPC) monolayers adsorbed at a liquid−liquid interface were determined.

Simultaneous measurement of spectroscopic and physiological signals from a planar bilayer system: detecting voltage-dependent movement of a membrane-incorporated peptide.

We developed an experimental system that can measure spectroscopic and physiological signals simultaneously from ion channels in a planar lipid bilayer, to study the relationship between the structure and function of the ion channels. While the membrane potential was clamped, fluorescent emission and ionic currents were measured simultaneously. The fluorescent emissions from a planar bilayer constructed in a specially designed chamber were monitored exclusively, and the signal intensity was measured with a photon-counting system. The intensity of fluorescence and spectral shape were measured successfully from the planar bilayer, with a high signal-to-noise ratio. The system can measure the intensity of fluorescence from a restricted area of the planar bilayer, with a diameter of 70 micrometer and a focal depth of 15 micrometer. The low background signal was achieved by optimizing the optical system. More than 95% of the measured fluorescence comes from the planar lipid bilayer. A 22-mer peptide with a sequence identical to that of the S4 segment of the electric eel sodium channel domain IV was synthesized and fluorescence-labeled. This peptide formed a voltage-dependent ion channel in a planar bilayer. The changes in the intensity of the fluorescence accompanying ionic currents generated by a voltage clamp suggest that voltage gating involves the insertion of the N-terminal of the peptide into the membrane. The electrical and optical signals were measured with a gate time of 10 ms. This measurement enabled the detection of movement of the membrane-incorporated peptides with channel opening.

A novel method for incorporation of ion channels into a planar phospholipid bilayer which allows solution changes on a millisecond timescale.

We have developed a method of rapidly changing the solutions on one side of a planar phospholipid bilayer. Bilayers can be painted on glass pipettes of tip diameter > or = 50 microns. By modifying an established method for rapid exchange of solutions bathing excised membrane patches, solution changes can be made at the bilayer within 10 ms. After incorporation of channels into the bilayer, the bilayer is moved into one of two parallel streams of solution flowing from a length of double-barrelled glass theta tubing. Activation of a solenoid system rapidly moves the theta tubing so that the bilayer is in the flow of the adjacent solution. For various reasons, the single-channel gating mechanisms of many channels are studied in planar bilayer systems. The conventional bilayer technique only allows for steady-state single-channel gating to be monitored. This novel method now allows the effects of rapid changes in modulators of channels incorporated into planar phospholipid bilayers to be measured.