Sangbaek Choi

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.

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.

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.