Detection of Trpm8 and RyR2 Activity on a Low-Noise, Automated Planar Lipid Bilayer Platform

Abstract

The ability to measure the activity of ion channels at the single molecule level in isolated planar lipid bilayers (PLB) is a critical tool for understanding the mechanism of such proteins. This is particularly important in the light of the many physiologically important ion channels that have been recombinantly expressed and reconstituted in recent times. However, most current PLB techniques involving bilayers painted across apertures >100 μm suffer from high noise arising from the capacitance of the bilayer and are severely limited in bandwidth. High noise and low bandwidth makes it difficult to investigate the fast gating properties of certain channels, thereby requiring complicated patch-clamp experiments.We have previously reported on the development of a novel platform, Nanopatch, based on glass nanopore membranes (GNMs, 200-3000 nm radius pore size), for performing high bandwidth, low noise measurements of ion channels on robust PLBs. Here we show further development of the platform with lower noise achieved with quartz membranes (QNMs), automated bilayer formation, and an electronically controlled auto-toggle routine for automated protein insertion. We also demonstrate the successful membrane incorporation and characterization of two multimeric ion channel proteins: the Ryanodine Receptor (RyR2) and the Transient receptor potential melastatin 8 (Trpm8). RyR2 channels were incorporated through osmotic swelling induced fusion of vesicles prepared from the sarcoplasmic reticulum, while Trpm8 channels were directly incorporated from a detergent solubilized protein solution. These experiments demonstrate the possibility of performing automated PLB measurements with patch-clamp like noise performance for a wide variety of ion channels.