Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels

Daisuke Tadaki,Ken-Ichi Ishibashi,Shun Araki,Kohei Arata,Takeshi Ohori,Yuzuru Tozawa,Ayumi Hirano-Iwata,Hideaki Yamamoto,Miyu Yoshida,Teng Ma,Miki Kato,Michio Niwano,Daichi Yamaura,Ryusuke Miyata

doi:10.1038/s41598-017-17905-x

Abstract

The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.

Highlights

Cells, the building blocks of life, are encapsulated within cell membranes that regulate ion flow in and out of cells
When buffered hydrofluoric acid (BHF) was used as a SiO2 etchant, the SiO2 layer remained on the Si3N4 layer and a much thicker septum (~400 nm) was formed (Fig. 2(b))
We previously reported on the formation of mechanically stable solvent-free bilayer lipid membrane (BLM) in apertures that were fabricated in Si chips using HF as the etchant for Step (6) (Fig. 1(a))[22,34]

Summary

Introduction

The building blocks of life, are encapsulated within cell membranes that regulate ion flow in and out of cells. BLM reconstitution of ion channels is a protein-based method for measuring ion channel currents The advantage of this system is its versatility in terms of producing target channels, including those prepared by cell-free protein synthesis[9], in which no cell culturing. The reduced BLM area of such preparations makes it more difficult to incorporate ion channel proteins, especially those delivered by proteoliposomes[17,18] To address this issue, several groups have fabricated relatively large apertures (several tens of micrometers in diameter) with tapered-wall edges in attempts to form stable BLMs18–24. Combining the BLM system with cell-free protein synthesis was investigated using the wild-type human ether-a-go-go-related gene (hERG) potassium channels as a representative example

Methods

Results

Conclusion