Micro‐ and Nano‐Technologies for Lipid Bilayer‐Based Ion‐Channel Functional Assays

Ion channel proteins provide gated pores that allow ions to passively flow across cell membranes. Owing to their crucial roles in regulating transmembrane ion flow, ion channel proteins have attracted the attention of pharmaceutical investigators as drug targets for use in the studies of both therapeutics and side effects. In this review, we discuss the current technologies that are used in the formation of ion channel‐integrated bilayer lipid membranes (BLMs) in microfabricated devices as a potential platform for next‐generation drug screening systems. Advances in BLM fabrication methodology have allowed the preparation of BLMs in sophisticated formats, such as microfluidic, automated, and/or array systems, which can be combined with channel current recordings. A much more critical step is the integration of the target channels into BLMs. Current technologies for the functional reconstitution of ion channel proteins are presented and discussed. Finally, the remaining issues of the BLM‐based methods for recording ion channel activities and their potential applications as drug screening systems are discussed.     

Synthetic Channel Specifically Inserts into the Lipid Bilayer of Gram‐Positive Bacteria but not that of Mammalian Erythrocytes

A series of tubular molecules with different lengths have been synthesized by attaching Trp‐incorporated peptides to the pillar[5]arene backbone. The tubular molecules are able to insert into the lipid bilayer to form unimolecular transmembrane channels. One of the channels has been revealed to specifically insert into the bilayer of the Gram‐positive bacteria. In contrast, this channel cannot insert into the membranes of the mammalian rat erythrocytes even at the high concentration of 100 μm . It was further demonstrated that, as a result of this high membrane selectivity, the channel exhibits efficient antimicrobial activity for the Gram‐positive bacteria and very low hemolytic toxicity for mammalian erythrocytes.     

Small‐Molecule Lipid‐Bilayer Anion Transporters for Biological Applications

The development of small‐molecule lipid‐bilayer anion transporters for potential future use in channel replacement therapy for the treatment of diseases caused by dysregulation of anion transport (such as cystic fibrosis), and in treating cancer by perturbing chemical gradients within cells, thus triggering apoptosis, is an area of intense current interest. This Minireview looks at recent developments in the design of small‐molecule transmembrane anion transporters and focuses on the progress so far in employing these compounds in biological systems.     

A New Cells‐Compatible Microfluidic Device for Single Channel Recordings

We report the fabrication of a microfluidic apparatus and the realization of a sensors based on PEDOT : PSS, a biocompatible semiconductor polymer used in substitution of standard electrodes for electrophysiological studies and for detection of nanopores in membrane. This gives the possibility to study the mechanisms of ions balance and molecular transport though cell membranes. In particular the apparatus is based on two chambers connected through an aperture in a PTFE sheet where lipid bilayer are formed using Montal‐Mueller method, and the pore‐forming proteins activity is detected by polymeric electrodes. This methodology could be applied to examine different membrane proteins for the purpose of biosensing, drug screening and nanopore technologies.     

Ion‐Channel Gating in Transmembrane Receptor Proteins: Functional Activity in Tethered Lipid Membranes

Through thiolipids a planar lipid bilayer (1) was immobilized on a gold support (2) for use as an electrode. This allows the detection of the ligand‐gating function of the natural transmembrane channel protein OmpF (3) reconstituted in the artificial membrane: the binding of a domain (4) of the toxin colicin N, observed by surface plasmon resonance, induces the blocking of the OmpF channel protein, as shown by impedance spectroscopy.     

Self‐Assembled Aggregates and Molecular Bilayer Films of a Double‐Chain Fullerene Lipid: Structure and Electrochemistry

The self‐aggregation behavior of C₆₀ fullerenes that bear two octadecyl chains (lipid 1 ) as well as the structures and electrochemical properties of cast films of 1 are described. We also examined the self‐aggregation behavior in organic solvents of three previously reported compounds: C₆₀ with three each of hexadecyl (lipid 2 ), tetradecyl (lipid 3 ), or dodecyl (lipid 4 ) chains. The fullerene lipids in alcohols spontaneously formed spherical aggregates, whose diameters are related to the alkyl‐chain lengths, concentrations of the fullerene lipids, and the solvent polarity. The morphologies of the aggregates showed temperature dependence. Cast films of 1 formed multimolecular bilayer structures that undergo a phase transition typical of lipid bilayer membranes. The electrochemistry of cast films of 1 on an electrode in aqueous medium exhibits temperature dependence.     

Biomimetic Transmembrane Channels with High Stability and Transporting Efficiency from Helically Folded Macromolecules

Membrane channels span the cellular lipid bilayers to transport ions and molecules into cells with sophisticated properties including high efficiency and selectivity. It is of particular biological importance in developing biomimetic transmembrane channels with unique functions by means of chemically synthetic strategies. An artificial unimolecular transmembrane channel using pore‐containing helical macromolecules is reported. The self‐folding, shape‐persistent, pore‐containing helical macromolecules are able to span the lipid bilayer, and thus result in extraordinary channel stability and high transporting efficiency for protons and cations. The lifetime of this artificial unimolecular channel in the lipid bilayer membrane is impressively long, rivaling those of natural protein channels. Natural channel mimics designed by helically folded polymeric scaffolds will display robust and versatile transport‐related properties at single‐molecule level.     

Dendritic folate rosettes as ion channels in lipid bilayers

The self-assembly of folate dendrimers into pi-stacked supramolecular rosettes is shown to produce ion channels in planar and spherical lipid bilayer membranes. The found ion channels are small, quite homogeneous, long-lived, ohmic, cation selective (Eisenman I), and blockable by the permeant cation.     

Self‐Assembled Columnar Triazole Quartets: An Example of Synergistic Hydrogen‐Bonding/Anion–π Interactions

The self‐assembly of triazole amphiphiles was examined in solution, the solid state, and in bilayer membranes. Single‐crystal X‐ray diffraction experiments show that stacked protonated triazole quartets (T₄) are stabilized by multiple strong interactions with two anions. Hydrogen bonding/ion pairing of the anions are combined with anion–π recognition to produce columnar architectures. In bilayer membranes, low transport activity is observed when the T₄ channels are operated as H⁺/X^? translocators, but higher transport activity is observed for X^? in the presence of the K⁺‐carrier valinomycin. These self‐assembled superstructures, presenting intriguing structural behaviors such as directionality, and strong anion encapsulation by hydrogen bonding supported by vicinal anion–π interactions can serve as artificial supramolecular channels for transporting anions across lipid bilayer membranes.     

Electrophysiological Characterization of Transport Across Outer‐Membrane Channels from Gram‐Negative Bacteria in Presence of Lipopolysaccharides

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we present an approach using fusion of native outer membrane vesicles (OMVs) into a planar lipid bilayer, allowing characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion surprisingly revealed only single or few channel activities. The asymmetry of the OMVs translates after fusion into the lipid membrane with the lipopolysaccharides (LPS) dominantly present at the side of OMV addition. Compared to the conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles for functional and structural studies of membrane channels in the native membrane.     

Potential analytical applications of lysenin channels for detection of multivalent ions

Transmembrane protein transporters possessing binding sites for ions, toxins, pharmaceutical drugs, and other molecules constitute excellent candidates for developing sensitive and selective biosensing devices. Their attractiveness for analytical purposes is enhanced by the intrinsic amplification capabilities shown when the binding event leads to major changes in the transportation of ions or molecules other than the analyte itself. The large-scale implementation of such transmembrane proteins in biosensing devices is limited by the difficulties encountered in inserting functional transporters into artificial bilayer lipid membranes and by the limitations in understanding and exploiting the changes induced by the interaction with the analyte for sensing purposes. Here, we show that lysenin, a pore-forming toxin extracted from earthworm Eisenia foetida, which inserts stable and large conductance channels into artificial bilayer lipid membranes, functions as a multivalent ion-sensing device. The analytical response consists of concentration and ionic-species-dependent macroscopic conductance inhibition most probably linked to a ligand-induced gating mechanism. Multivalent ion removal by chelation or precipitation restores, in most cases, the initial conductance and demonstrates reversibility. Changes in lipid bilayer membrane compositions leading to the absence of voltage-induced gating do not affect the analytical response to multivalent ions. Microscopic current analysis performed on individual lysenin channels in the presence of Cu²⁺ revealed complex open–closed transitions characterized by unstable intermediate sub-conducting states. Lysenin channels provide an analytical tool with a built-in sensing mechanism for inorganic and organic multivalent ions, and the excellent stability in an artificial environment recommend lysenin as a potential candidate for single-molecule detection and analysis.     

Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5－Ditetradecyl－2－ （2－trimethyl－ammonioethyl）－l, 3－dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-l,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane.The voltammetric study showed that the lipid membrane can open ion channels in the presence of ClO4^- stimulant with Ru(bpy)3^2 as marker ions and give distinct channel currents.The channels can be dosed and open up again many times by removing or introducing ClO4^- anions.     

Highly conducting transmembrane pores formed by aromatic oligoamide macrocycles

Oligoamide macrocycles 1d and 1e, which carry membrane-compatible side chains and contain a hydrophilic, noncollapsible cavity, were found to mediate high ion flux across a lipid bilayer, as demonstrated by results from (23)Na NMR and planar bilayer conductance measurements. The measured transmembrane single channel currents are very high, rivaling those typically associated with pore-forming protein toxins. The obtained results have demonstrated the promise of developing large, highly conducting channels based on nanopores formed by oligoamide macrocycles.     

Blockable Zn10L15 Ion Channels through Subcomponent Self‐Assembly

Metal–organic anion channels based on Zn₁₀L₁₅ pentagonal prisms have been prepared by subcomponent self‐assembly. The insertion of these prisms into lipid membranes was investigated by ion‐current and fluorescence measurements. The channels were found to mediate the transport of Cl⁻ anions through planar lipid bilayers and into vesicles. Tosylate anions were observed to bind and plug the central channels of the prisms in the solid state and in solution. In membranes, dodecyl sulfate blocked chloride transport through the central channel. Our Zn₁₀L₁₅ prism thus inserts into lipid bilayers to turn on anion transport, which can then be turned off through addition of the blocker dodecyl sulfate.     

Heavy‐Atom Labeled Transmembrane β‐Peptides: Synthesis,CD‐Spectroscopy,and X‐ray Diffraction Studies in Model Lipid Multilayer

Transmembrane β‐peptides are promising candidates for the design of well‐controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β‐peptides with and without tryptophan anchors, as well as a novel iodine‐labeled d ‐β³‐amino acid. By using one or more of the heavy‐atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron‐density profile determined by X‐ray reflectivity. The β‐peptides were synthesized through manual Fmoc‐based solid‐phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right‐handed 3₁₄‐helix secondary structure, as shown by circular dichroism spectroscopy. We then integrated the β‐peptide into solid‐supported membrane stacks and carried out X‐ray reflectivity and grazing incidence small‐angle X‐ray scattering to determine the β‐peptide orientation and its effect on the membrane bilayers. These β‐peptides adopt a well‐ordered transmembrane motif in the solid‐supported model membrane, maintaining the basic structure of the original bilayer with some distinct alterations. Notably, the helical tilt angle, which accommodates the positive hydrophobic mismatch, induces a tilt of the acyl chains. The tilted chains, in turn, lead to a membrane thinning effect.     

Voltage‐Driven Reversible Insertion into and Leaving from a Lipid Bilayer: Tuning Transmembrane Transport of Artificial Channels

Three new artificial transmembrane channel molecules have been designed and synthesized by attaching positively charged Arg‐incorporated tripeptide chains to pillar[5]arene. Fluorescent and patch‐clamp experiments revealed that voltage can drive the molecules to insert into and leave from a lipid bilayer and thus switch on and off the transport of K⁺ ions. One of the molecules was found to display antimicrobial activity toward Bacillus subtilis with half maximal inhibitory concentration (IC₅₀) of 10 μM which is comparable to that of natural channel‐forming peptide alamethicin.     

Reconstitution of the Torpedo californica nicotinic acetylcholine receptor into planar lipid bilayers

Detergent-solubilized acetylcholine receptor (nAcChR) proteins can be purified by affinity chromatography and reconstituted into lipid vesicles and afterwards into planar lipid bilayer membranes via, in principle, two methods: fusion or assembly from two vesicle-spread monolayers. In the presence of agonists (carbamoylcholine, suberoyldicholine) different kinds of channel openings are recorded: fast single channels, bursts and long openings with short closures in between. Similar results have been obtained with reconstituted membrane fragments rich in nAcChR. In addition, Torpedo californica nAcChR proteins give rise to fuzzy channels and less defined events of conductivity, which “reemerge” again all the time. Frequently the channel events have conductance levels of about 200 to 300 pS, obviously simultaneous openings of several aggregated receptors. Under these conditions 40 pS conductance events occur also. It appears that the conductance of the channels measured is a multiple of 6.3 pS. Often, with the same sample, no channel openings are seen. Contrary to patch-clamp investigations on whole cells, AcChR-channel openings in reconstituted systems occur only several minutes after agonist application and not immediately. It is not clear whether the “reconstituted channels” reflect rapid activation or whether they result from desensitized receptor states only. Although a clear-cut correlation of channel event and channel protein unit is only possible by reconstitution of the biochemically characterized protein, e.g. monomer, dimer or higher oligomers, the reconstitution technique is still in its infancy.     

Synthetic ion channel activity documented by electrophysiological methods in living cells

Hydraphiles are synthetic ion channels that use crown ethers as entry portals and that span phospholipid bilayer membranes. Proton and sodium cation transport by these compounds has been demonstrated in liposomes and planar bilayers. In the present work, whole cell patch clamp experiments show that hydraphiles integrate into the membranes of human embryonic kidney (HEK 293) cells and significantly increase membrane conductance. The altered membrane permeability is reversible, and the cells under study remain vital during the experiment. Control compounds that are too short (C(8)-benzyl channel) to span the bilayer or are inactive owing to a deficiency in the central relay do not induce similar conductance increases. Control experiments confirm that the inactive channel analogues do not show nonspecific effects such as activation of native channels. These studies show that the combination of structural features that have been designed into the hydraphiles afford true, albeit simple, channel function in live cells.     

Facilitated Transport of Ions and Glucose by Amphotericin B Across Lipid Bilayers in the Presence or Absence of Cholesterol

The transport of ions and glucose across bilayer lipid membranes (BLM) facilitated by amphotericin B (AmB) is studied by use of planar BLMs and liposomal membranes. The transport characteristics change with time in the presence of cholesterol, while it is independent of time in the absence of cholesterol. The carrier‐type transport is observed immediately after the addition of AmB. In the presence of cholesterol, AmB forms a 1 : 1 complex with cholesterol and the channel is formed by aggregation of AmB‐cholesterol complexes. It is concluded that the number of the channels increases with time and that the carrier‐type transport decreases instead.