Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes

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Abstract
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. Significance We present a method for measuring the conductance of ion channels at bandwidths up to 500 kHz by fabricating lipid membranes directly on the surface of a custom amplifier chip. We apply this approach to the RyR1 receptor, enabling us to identify additional closed states for calcium-dependent inactivation at microsecond temporal resolutions. Additional data analysis using extended beta distributions allows us to detect gating events as short as 35 ns, a timescale that approaches that of single-file ion translocation. These measurement techniques hold the promise of reaching timescales for studying the kinetics of ion channels, achievable now only with computer-based molecular dynamics simulations.
Funding Information
  • HHS | NIH | National Human Genome Research Institute (R01HG009189)
  • HHS | NIH | National Human Genome Research Institute (R01HG006879)
  • HHS | NIH | National Institute of General Medical Sciences (P41GM116799)
  • HHS | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR060037)
  • HHS | NIH | National Heart, Lung, and Blood Institute (R01HL061503)
  • HHS | NIH | National Heart, Lung, and Blood Institute (T32HL120826)

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