Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Wavelet Denoising of High-Bandwidth Nanopore and Ion-Channel Signals.

Recent work has pushed the noise-limited bandwidths of solid-state nanopore conductance recordings to more than 5 MHz and of ion channel conductance recordings to more than 500 kHz through the use of integrated complementary metal-oxide-semiconductor (CMOS) integrated circuits. Despite the spectral spread of the pulse-like signals that characterize these recordings when a sinusoidal basis is employed, Bessel filters are commonly used to denoise these signals to acceptable signal-to-noise ratios (SNRs) at the cost of losing many of the faster temporal features. Here, we report improvements to the SNR that can be achieved using wavelet denoising instead of Bessel filtering. When combined with state-of-the-art high-bandwidth CMOS recording instrumentation, we can reduce baseline noise levels by over a factor of 4 compared to a 2.5 MHz Bessel filter while retaining transient properties in the signal comparable to this filter bandwidth. Similarly, for ion-channel recordings, we achieve a temporal response better than a 100 kHz Bessel filter with a noise level comparable to that achievable with a 25 kHz Bessel filter. Improvements in SNR can be used to achieve robust statistical analyses of these recordings, which may provide important insights into nanopore translocation dynamics and mechanisms of ion-channel function.

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

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.