Parallel Recordings of Transmembrane hERG Channel Currents Based on Solvent-Free Lipid Bilayer Microarray

Abstract

The reconstitution of ion-channel proteins in artificially formed bilayer lipid membranes (BLMs) forms a well-defined system for the functional analysis of ion channels and screening of the effects of drugs that act on these proteins. To improve the efficiency of the BLM reconstitution system, we report on a microarray of stable solvent-free BLMs formed in microfabricated silicon (Si) chips, where micro-apertures with well-defined nano- and micro-tapered edges were fabricated. Sixteen micro-wells were manufactured in a chamber made of Teflon^®, and the Si chips were individually embedded in the respective wells as a recording site. Typically, 11 to 16 BLMs were simultaneously formed with an average BLM number of 13.1, which corresponded to a formation probability of 82%. Parallel recordings of ion-channel activities from multiple BLMs were successfully demonstrated using the human ether-a-go-go-related gene (hERG) potassium channel, of which the relation to arrhythmic side effects following drug treatment is well recognized.

Keywords: bilayer lipid membrane (BLM); human ether-a-go-go-related gene (hERG) channel; ion channel; microarray.

Figure 1

(a) Schematic drawing of single Si chip. (upper) Top view from the Si₃N₄ side. (middle) Cross-sectional side view. (bottom) Field emission scanning electron microscopy (FE-SEM) image around the edge region of the micro-aperture. Tilt angle: 65°. (b) Schematic drawing of chip holders and Si chips. (c) Schematic drawing of the chamber system that is held together with polyetheretherketone (PEEK) screws. (d) Photograph of chip holders A and B. Sixteen Si chips are placed in chip holder A. (e) Photograph of the components used to assemble the system.

Figure 2

Schematic images of procedures for simultaneous formation of bilayer lipid membranes (BLMs) and rearrangement for multiarray measurements.

Figure 3

(a) Schematic drawing of the electrode holder. (b) Photographs of the electrode holder before and after inserting 16 Ag/AgCl electrodes.

Figure 4

Electrical current flowing between two Ag/AgCl electrodes inserted in the two compartments, as shown in Figure 2. Applied voltage was 0 mV. Shaded regions represent expected times when buffer levels were gradually passing each row of the four wells. Schematic diagrams of the relationship between the buffer levels and the position of the wells corresponding to the respective shaded times are also shown.

Figure 5

An example of simultaneously recorded transmembrane currents from the 15 BLMs, when the applied voltage was repeatedly switched from 0 to +1 V, from +1 to 0 V, from 0 to −1 V, and from −1 to 0 V.

Figure 6

An example trace of the human ether-a-go-go-related gene (hERG) single-channel current (middle) recorded by applying a repetitive voltage-step protocol: −100 mV for 3.1 s after a 300 ms prepulse of +50 mV (upper). The enlarged current waveform is also displayed (bottom). The signals were recorded with a 1 kHz low-pass filter at a sampling frequency of 20 kHz. The current trace was filtered offline at a cutoff frequency of 0.7 kHz.

Figure 7

Parallel recordings of the hERG channel activities before and after the addition of E-4031. The voltage protocol shown in Figure 6 was used to elicit channel activities. For Ch. 1 and 13, current traces during the applied voltage kept at −100 mV are shown. For the current trace shown in Ch. 16, the applied voltage was switched from −100 to +50 mV and then switched back to −100 mV. E-4031 was added to the common side solution to give a final concentration of 10 μM. The signals were recorded with a 1 kHz low-pass filter at a sampling frequency of 20 kHz. The currents were filtered offline at a cutoff frequency of 0.7 kHz.