Andreas J. W. Hartel, Peijie Ong, Indra Schroeder, M. Hunter Giese, Siddharth Shekar, Oliver B. Clarke, Ran Zalk, Andrew R. Marks, Wayne A. Hendrickson and Kenneth L. Shepard. Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes. PNAS February 20, 2018. 115 (8) E1789-E1798, DOI:10.1073/pnas.1712313115.
Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.