This paper reports on the reconstitution of human ether-a-go-go-related gene (hERG) channels in artificial bilayer lipid membranes (BLMs) formed in micropores fabricated in silicon chips. The hERG channels were isolated from Chinese hamster ovary cell lines expressing the channels and incorporated into the BLMs formed by a process in which the two lipid monolayers were folded into the micropores. The characteristic features of hERG channels reported by the patch-clamp method, including single-channel conductance, voltage dependence, sensitivity to typical drugs and dependence on the potassium concentration, were investigated in the BLM reconstitution system. The BLM with hERG channels incorporated exhibited a lifetime of ∼65 h and a tolerance to repetitive solution exchanges. Such stable BLMs containing biological channels have the potential for use in a variety of applications, including high-throughput drug screening for various ion-channel proteins.

Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Na v 1.5), and the human GABA A receptor (GABA A R) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 Â g). The characteristic channel properties were retained for hERG, Na v 1.5, and GABA A R channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.

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.

In this paper, we will discuss our recent approaches for the formation of mechanically stable artificial bilayer lipid membranes (BLMs) by combining with silicon (Si) micro-fabrication techniques and their application to recording activities of biological channels. BLMs were prepared across microfabricated pores in thin Si3N4 septa of Si chips. The edge of the pores was smoothly tapered in nanometer range, which was useful for stabilizing the BLMs suspended in the pores. The BLMs showed a membrane lifetime of＞40 h, tolerance to a high voltage of±1 V, and tolerance to repetitive solution exchanges. Application to a platform for recording biological channels has been examined by using the human ether-a-go-go-related gene (hERG) potassium channel as an illustrative example. Such stable BLMs with integrated biological channels have the potential for use in a variety of applications, including high-throughput drug screening for various ion-channel proteins.