Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment

Abstract

Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca²⁺ release channels that are responsible for the increase of cytosolic Ca²⁺ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca²⁺ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.

Keywords: Amphipol; Cryospecimen preparation; Detergents; Electron cryo-microscopy; Membrane proteins; Ryanodine receptor.

Fig 1.

Timeline summary of RyR1 structural studies. RyR1 was first identified as large electron-dense “foot” observed between the junctions of T-tubule and SR membranes. Over the next two decades many efforts were made to molecularly identify the structure observed in the triad junctions and its role in muscle physiology. Eventually, through the use of H-ryanodine, differential centrifugation and biophysical characterizations, RyR1 was solidified as the intracellular Ca²⁺ release channel responsible for the release of Ca²⁺ preceding muscle contraction. Due to its relative ease of purification and large size, the structure of RyR1 was investigated by single-particle electron-microscopy. The first 3D structure was obtained by negative stain microscopy, reveling basic morphological features, albeit in a stained and dehydrated state. The first depictions of RyR1 in the more native, hydrated conditions came ~4 years later by cryo-EM and were solved to ~30 Å resolution.^, Low-resolution structural dynamics of RyR1 gating were described by adding ligands that affect channel open probability to the cryospecimen prior to virtification.^, Several additional low-resolution structures of RyR1 were solved that localized small molecule binding sites (CaM, FKBP12 and imperatoxin) and functional domains on the 3D structure of RyR1.^,, Structures of RyR2 and RyR3 isoforms were also determined by cryo-EM and appear similar in nature to RyR1. ^, In two decades since the first structure of RyR1 by cryo-EM was determined, ~1 nm resolution structures were obtained and density based models of channel gating were proposed.^, Homology models for the N-terminal domain of the channel were created based on structures of the IPR1 N-terminus and computationally fitted to the map ^, Several secondary structure elements in the cytoplasmic and transmembrane domains and subunit boundaries were detectable resulting in a molecular model for some transmembrane helices. Gating induced structural changes were investigated in a ~1 nm resolution structure. Structural models for three disease hot-spot domains were determined by X-ray crystallography: the RyR1 N-terminal domain (residues 1-559; PDB: 2XOA), phosphorylation domain (residues 2734-2940; PDB: 4ERT) and SPRY2 domain (residues 1070-1246; PDB: 4P9I, 4P9J, 4P9L).^,,, With state of the art imaging technology in place, the future for the Ca²⁺ release channel is ripe to proceed towards near-atomic resolutions.

Fig 2.

3D structures of the tetrameric RyR1 channel determined by cryo-EM. Surface representations of RyR1 density maps by single-particle cryo-EM viewed in three orthogonal views – from cytoplasm (top), along the membrane plane (middle) and from luminal side (bottom) of the membrane. Left to right are RyR1 density maps at 14 Å (EMD-1274), 10.2 Å (EMD-5014) and 9.6 Å (EMD-1275) resolutions.^,,

Fig 3.

Cryo-EM images of ice-embedded purified RyR1: in the presence of 0.4 % CHAPS (A) in the presence of A8-35 (B); in the presence of A8-35/n-octyl glucoside [OG]. Note preferred orientation of RyR1 particles in (B), while the particles are randomly oriented within the vitreous ice due to achieved optimal protein/Apol8-35/OG ratio in the cryospecimen shown in (C). Images were recorded on a Gatan 4k x 4k CCD camera using JEM2010F cryomicroscope operated under minimal electron dose conditions (~20 ^e-/Å). Scale bars are 500 Å.

Fig 4.

Scatchard analysis of [H]-ryanodine binding to RyR1: in skeletal muscle SR membranes (•), purified RyR1 bound to CHAPS (▲) and 800 purified RyR1 in complex with A8-35 (■). Linear fitting yielded Kd of 1.99 nM and Bmax of 60.4 pmol/mg of protein for RyR1/A8-35, and Kd of 41.27 nM and Bmax of 3.07 pmol/mg for RyR1/CHAPS and Kd of 2.54 nM and Bmax of 27.9 pmol/mg for SR membranes in high Ca²⁺ conditions (200μM Ca²⁺), indicating that the high-affinity binding site for ryanodine is retained in RyR1/Apol and similar to that of RyR1 embedded within the SR membrane.