Incorporation of RyR2 and Other Ion Channels into Nanopore Based Planar Lipid Bilayers for Low Noise Single Channel Recordings

Abstract

Measurement of ion channel activity at the single molecule level in isolated planar lipid bilayers (PLB) is a critical biophysical tool for understanding the function of such proteins. However, current PLB techniques involving bilayers painted across apertures >100 μm suffer from high noise arising from the capacitance of the bilayer and is severely limited in bandwidth. Therefore, it is difficult to investigate the fast gating properties of certain channels e.g the Ryanodine Receptor (RyR2). RyR2 channels, which play a key role in the intracellular Ca2+ induced calcium release mechanism, demonstrate a complex gating characterized by bursts of very fast open-close transitions that cannot be resolved by conventional PLB apparatus. Furthermore, the low current amplitude these channels produce in presence of the physiological ion Ca2+, complicates the analysis.We have developed a robust platform based on glass or quartz nanopore membranes (GNMs, 200-3000 nm radius pore size), for performing high bandwidth, low noise measurements of such ion channels in lipid bilayers. Previously, we have demonstrated incorporation of bacterial toxins [1] and porins in these small bilayers. Here we report the successful incorporation and measurement of RyR2 activity in such a system. Vesicles prepared from sarcoplasmic reticulum enriched in RyR2 channels were fused through osmotic swelling to PLBs formed on a GNM with a 3000 nm radius orifice, allowing unprecedented resolution of single RyR2 channel events at 10 kHz.In addition, we have fused vesicles containing nystatin and ergosterol to small PLBs (< 1000nm radius) successfully. The latter method has the potential to provide a general technique for incorporation of a variety of ion channels in small GNM bilayers.[1] White et al J. Am. Chem. Soc., 129, 11766-11775, 2007