Automated Parallel Recordings of Topologically Identified Single Ion Channels

Abstract

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Channel Current Analysis for Pore-forming Properties of an Antimicrobial Peptide, Magainin 1, Using the Droplet Contact Method

“…11,15 Four individual BLMs can be formed simultaneously in this device, which allowed for a higher-throughput measurement compared to the conventional system. Using this device, the DOPC or DOPE/DOPG (3:1 mol/mol) mixture (lipids/n-decane, 10 mg/ mL) solution (2.3 μL) was poured into each chamber.…”

“…11 A planar lipid bilayer can be spontaneously formed at the interface between contacting microdroplets in an oil/lipid mixture solution. 12,13 In this method, membrane stability is improved by reducing the contact area.…”

“…11 Using bilayer lipid membranes (BLMs), we have been measuring various membrane protein and biological nanopores, e.g., single molecule detection using DNA aptamers, 14 high-throughput ion channel measurements at the single-protein level, 11,15 solution-exchange experiment in the microdroplet system, 16 and the development of portable nanopore sensors. 17 In this paper, we report on the pore-forming properties of magainin 1 (GIGKFLHSAGKFGKAFVGEIMKS) from channel current conductance measurements using the high-throughput DCM.…”

Hairpin DNA Unzipping Analysis Using a Biological Nanopore Array

“…12 Furthermore, we constructed a droplet array for high-throughput measurements. 13 Using bilayer lipid membranes (BLMs), we measured various membrane proteins and biological nanopore, for example, single molecule detection using DNA aptamers, 14 high-throughput ion channel measurements at the single-protein level, 13,15,16 solutionexchange techniques via the microdroplet system, 17 and the development of portable nanopore sensors. 18 In this study, we Figure 1.…”

Scalable micro-cavity bilayer lipid membrane arrays for parallel ion channel recording

“…Recently it was demonstrated that the stability of artificial bilayer membranes can be significantly increased by using shaped apertures fabricated in negative resists such as SU8 [7], or complex (incline/rotation) exposure technique [8]. In order to increase throughput, bilayer arrays have been developed, for example using stereo-lithography to define chambers around a thin film of a hydrophobic polymer that includes an aperture for bilayer formation [9,10]. Both ease of bilayer formation and stability has been improved through the use of small cavities in the range 6-50 m diameter [11][12][13].…”