Tentonin 3 is a pore-forming subunit of a slow inactivation mechanosensitive channel.

Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.

Control of artificial membrane fusion in physiological ionic solutions beyond the limits of electroformation.

Membrane fusion, merging two lipid bilayers, is crucial for fabricating artificial membrane structures. Over the past 40 years, in contrast to precise and controllable membrane fusion in-vivo through specific molecules such as SNAREs, controlling the fusion in-vitro while fabricating artificial membrane structures in physiological ionic solutions without fusion proteins has been a challenge, becoming a significant obstacle to practical applications. We present an approach consisting of an electric field and a few kPa hydraulic pressure as an additional variable to physically control the fusion, enabling tuning of the shape and size of the 3D freestanding lipid bilayers in physiological ionic solutions. Mechanical model analysis reveals that pressure-induced parallel/normal tensions enhance fusion among membranes in the microwell. In-vitro peptide-membrane assay, mimicking vesicular transport via pressure-assisted fusion, and stability of 38 days with in-chip pressure control via pore size-regulated hydrogel highlight the potential for diverse biological applications.

Quantitative Analysis of the Membrane Affinity of Local Anesthetics Using a Model Cell Membrane.

Local anesthesia is a drug that penetrates the nerve cell membrane and binds to the voltage gate sodium channel, inhibiting the membrane potential and neurotransmission. It is mainly used in clinical uses to address the pain of surgical procedures in the local area. Local anesthetics (LAs), however, can be incorporated into the membrane, reducing the thermal stability of the membrane as well as altering membrane properties such as fluidity, permeability, and lipid packing order. The effects of LAs on the membrane are not yet fully understood, despite a number of previous studies. In particular, it is necessary to analyze which is the more dominant factor, the membrane affinity or the structural perturbation of the membrane. To analyze the effects of LAs on the cell membrane and compare the results with those from model membranes, morphological analysis and 50% inhibitory concentration (IC50) measurement of CCD-1064sk (fibroblast, human skin) membranes were carried out for lidocaine (LDC) and tetracaine (TTC), the most popular LAs in clinical use. Furthermore, the membrane affinity of the LAs was quantitatively analyzed using a colorimetric polydiacetylene assay, where the color shift represents their distribution in the membrane. Further, to confirm the membrane affinity and structural effects of the membranes, we performed an electrophysiological study using a model protein (gramicidin A, gA) and measured the channel lifetime of the model protein on the free-standing lipid bilayer according to the concentration of each LA. Our results show that when LAs interact with cell membranes, membrane affinity is a more dominant factor than steric or conformational effects of the membrane.

Multilayered film for the controlled formation of freestanding lipid bilayers.

A freestanding lipid bilayer or black lipid membrane is a powerful tool for studying ion channels and for biophysical studies of other membrane proteins under controlled chemical and physical conditions. Even though the lipid bilayer has been considered an excellent sensing platform to detect diverse single molecules from nucleotides to cells, it is not yet widely used, mainly due to its low stability and the expertise needed for membrane formation. To ameliorate the issues of conventional membrane formation techniques, we report a novel layered film that consists of a nonporous layer sandwiched between two porous layers to facilitate bilayer formation. Moreover, the absorption of excess solvent present in the membrane precursor solution can be achieved by the film, enabling control over the membrane formation process. Through this layered design, we could obtain an ideal film that has a reduced and controlled membrane formation time (<30 min) and a sufficient bilayer lifetime (3 h) for ion channel studies and biosensing.

Aptamer-Conjugated Polydiacetylene Colorimetric Paper Chip for the Detection of Bacillus thuringiensis Spores.

A colorimetric polydiacetylene (PDA) paper strip sensor that can specifically recognize Bacillus thuringiensis (BT) HD-73 spores is described in this work. The target-specific aptamer was combined with PDA, and the aptamer-conjugated PDA vesicles were then coated on polyvinylidene fluoride (PVDF) paper strips by a simple solvent evaporation method. The PDA-aptamer paper strips can be used to detect the target without any pre-treatment. Using the paper strip, the presence of BT spores is directly observable by the naked eye based on the unique blue-to-red color transition of the PDA. Quantitative studies using the paper strip were also carried out by analyzing the color transitions of the PDA. The specificity of this PDA sensor was verified with a high concentration of Escherichia coli, and no discernable change was observed. The observable color change in the paper strip occurs in less than 1 h, and the limit of detection is 3 × 107 CFU/mL, much below the level harmful to humans. The PDA-based paper sensor, developed in this work, does not require a separate power or detection device, making the sensor strip highly transportable and suitable for spore analysis anytime and anywhere. Moreover, this paper sensor platform is easily fabricated, can be adapted to other targets, is highly portable, and is highly specific for the detection of BT spores.

Synthetic Anion Transporters as Endoplasmic Reticulum (ER) Stress Inducers.

Cl--ion transporters (2a-2h) were synthesized based on the binding motifs of prodigiosin. Transporter 2e clearly displays Cl--ion transportation activity across both model and live cell membranes. Furthermore, 2e can disrupt Ca2+ homeostasis and increase the intracellular concentration of Ca2+ in the DLD-1 cell. This disruption can lead to Caspase-dependent apoptosis supported by CHOP expression (a marker of ER stress) and the appearance of the cleaved forms of Caspase 3 and PARP.

A Helical Polypeptide-Based Potassium Ionophore Induces Endoplasmic Reticulum Stress-Mediated Apoptosis by Perturbing Ion Homeostasis.

Perturbation of potassium homeostasis can affect various cell functions and lead to the onset of programmed cell death. Although ionophores have been intensively used as an ion homeostasis disturber, the mechanisms of cell death are unclear and the bioapplicability is limited. In this study, helical polypeptide-based potassium ionophores are developed to induce endoplasmic reticulum (ER) stress-mediated apoptosis. The polypeptide-based potassium ionophores disturb ion homeostasis and then induce prolonged ER stress in the cells. The ER stress results in oxidative environments that accelerate the activation of mitochondria-dependent apoptosis. Moreover, ER stress-mediated apoptosis is triggered in a tumor-bearing mouse model that suppresses tumor proliferation. This study provides the first evidence showing that helical polypeptide-based potassium ionophores trigger ER stress-mediated apoptosis by perturbation of potassium homeostasis.

Biomimetic Membranes with Transmembrane Proteins: State-of-the-Art in Transmembrane Protein Applications.

In biological cells, membrane proteins are the most crucial component for the maintenance of cell physiology and processes, including ion transportation, cell signaling, cell adhesion, and recognition of signal molecules. Therefore, researchers have proposed a number of membrane platforms to mimic the biological cell environment for transmembrane protein incorporation. The performance and selectivity of these transmembrane proteins based biomimetic platforms are far superior to those of traditional material platforms, but their lack of stability and scalability rule out their commercial presence. This review highlights the development of transmembrane protein-based biomimetic platforms for four major applications, which are biosensors, molecular interaction studies, energy harvesting, and water purification. We summarize the fundamental principles and recent progress in transmembrane protein biomimetic platforms for each application, discuss their limitations, and present future outlooks for industrial implementation.

Nanopore based detection of Bacillus thuringiensis HD-73 spores using aptamers and versatile DNA hairpins.

A versatile nanopore sensing platform to detect any aptamer using nanopores by designing DNA hairpins that are complementary to the aptamer is described. This platform can detect the presence of aptamer binding targets regardless of their size, which has been a major hurdle for nanopore detection systems. Moreover, the signal-to-noise ratio is increased by eliminating most of the unwanted substances from the sample via simple sample preparation steps. To detect Bacillus thuringiensis HD-73 spores using this sensing platform, DNA hairpins complementary to the target-specific aptamers were designed, and the hairpins were characterized using alpha-hemolysin nanopores after the reaction of spores and aptamers and subsequent reaction with the complementary DNA hairpins. The platform exhibited a detection limit as low as 1.2 × 101 CFU mL-1 and was compatible with a wide range of spore concentrations from 1.2 × 101 CFU mL-1 to 1.2 × 106 CFU mL-1 while it is still expandable to higher spore concentrations.

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 °C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of < 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

Investigation of Ion Channel Activities of Gramicidin A in the Presence of Ionic Liquids Using Model Cell Membranes.

Ionic liquids (ILs) are considered to be green solvents because of their non-volatility. Although ILs are relatively safe in the atmospheric environment, they may be toxic in other environments. Our previous research showed that the cytotoxicity of ILs to biological organisms is attributable to interference with cell membranes by IL insertion. However, the effects of ILs on ion channels, which play important roles in cell homeostasis, have not been comprehensively studied to date. In this work, we studied the interactions between ILs and lipid bilayer membranes with gramicidin A ion channels. We used two methods, namely electrical and fluorescence measurements of ions that permeate the membrane. The lifetimes of channels were increased by all the ILs tested in this work via stabilizing the compressed structure of the lipid bilayer and the rate of ion flux through gA channels was decreased by changing the membrane surface charge. The former effect, which increased the rate of ion flux, was dominant at high salt concentrations, whereas the latter, which decreased the rate of ion flux, was dominant at low salt concentrations. The effects of ILs increased with increasing concentration and alkyl chain length. The experimental results were further studied using molecular dynamics simulations.

Synthetic anion transporters that bear a terminal ethynyl group.

Cl(-) transporters that bear a terminal ethynyl group were synthesized; they consist of non-pyrrolic hydrogen bond motifs such as phenolic OH, amide NH, and triazole CH. The ethynyl group of these non-pyrrolic analogs plays an important role in chloride efflux and they exhibit no significant cytotoxic activity.

Automated lipid membrane formation using a polydimethylsiloxane film for ion channel measurements.

A black lipid membrane (BLM) is a powerful platform for studying the electrophysiology of cell membranes as well as transmembrane proteins. However, BLMs have disadvantages in terms of stability, accessibility, and transportability, which preclude their industrial applications. To resolve these issues, frozen membrane precursor (MP) was devised to improve the transportability and storability of BLMs. As described previously, MP is a storable and transportable platform that can be delivered to the point-of-use, where BLMs are automatically formed upon thawing at room temperature. However, MP has an inconsistent thinning-out time, ranging from 30 min to 24 h, as well as a low success rate of BLM formation (~27%), which make it undesirable for practical use. In our study, polydimethylsiloxane (PDMS) was introduced as a replacement for conventionally used Teflon film to control thinning-out time. As such, we used a PDMS thin-film, a porous-structured hydrophobic polymer, and squalene, a high viscosity solvent, to facilitate membrane formation, whereas the absorption rates of solvents were controlled to achieve consistent BLM formation time. We successfully reduced thinning-out time down to <1 h as well as enhanced the success rate of BLM formation to greater than 80%. Moreover, we demonstrated the feasibility of our platform for use in drug screening using gramicidin A and guanidine.

Synthetic biomimetic membranes and their sensor applications.

Synthetic biomimetic membranes provide biological environments to membrane proteins. By exploiting the central roles of biological membranes, it is possible to devise biosensors, drug delivery systems, and nanocontainers using a biomimetic membrane system integrated with functional proteins. Biomimetic membranes can be created with synthetic lipids or block copolymers. These amphiphilic lipids and polymers self-assemble in an aqueous solution either into planar membranes or into vesicles. Using various techniques developed to date, both planar membranes and vesicles can provide versatile and robust platforms for a number of applications. In particular, biomimetic membranes with modified lipids or functional proteins are promising platforms for biosensors. We review recent technologies used to create synthetic biomimetic membranes and their engineered sensors applications.