HERG Ion Channel Activity Measured in Planar Lipid Bilayer Arrays: Reconstitution from Eukaryotic Cell-Free Expression System and Cellular Membrane Preparations

Abstract

Ion channels are important pharmacological targets. Therefore there is an urgent need for new technologies for high throughput ion channel activity monitoring and pharmacology screening capable to substitute currently used labor-intensive and expensive cell-based electrophysiological assays. Ion channels reconstituted into artificial membrane arrays may serve as such test system offering a substantial simplification as well as great flexibility in controlling the ion channel environment and experimental conditions.Human ether-a-go-go-related gene (hERG), a voltage dependent potassium channel is a major target of pharmacology safety screening due to its critical importance in heart function. Here we report on parallel high-resolution electrophysiological recordings from different hERG-containing preparations reconstituted into bilayer lipid membranes on the micro electrode cavity array (MECA) chip.Two different sources of hERG channels have been investigated: (1) Membrane preparations from a hERG-expressing HEK293 cell line, and (2) Vesicles collected after cell-free expression of the hERG channels with their co-translational integration into microsomes or liposomes. The hERG-containing vesicles were fused with lipid bilayers preformed on the MECA chip. For both preparations fluctuating currents of different amplitudes have been recorded following the fusion. Single channel and channel ensemble currents depending on the vesicle preparation have been characterized. The recorded currents were shown to be partially inhibited by the hERG specific blockers E4031 and astemizole.High channel expression level as well as optimized vesicles preparation and fusion conditions were shown to be crucial for the reproducible channel recordings in the artificial membrane system. Addition of charged lipids (POPG/DOTAP) to the liposomes and acceptor membranes was found to facilitate vesicle fusion and improve the measurements success rate. The presented technology has a potential for a high throughput ion channels pharmacology screening in general.