The structural basis of ryanodine receptor ion channel function

Abstract

Large-conductance Ca²⁺ release channels known as ryanodine receptors (RyRs) mediate the release of Ca²⁺ from an intracellular membrane compartment, the endo/sarcoplasmic reticulum. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RyR3 is expressed at low levels in many tissues including brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four ∼12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases. RyRs share ∼70% sequence identity, with the greatest sequence similarity in the C-terminal region that forms the transmembrane, ion-conducting domain comprising ∼500 amino acids. The remaining ∼4,500 amino acids form the large regulatory cytoplasmic "foot" structure. Experimental evidence for Ca²⁺, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca²⁺ releasing agents caffeine and ryanodine. Recent work describing the near atomic structures of mammalian skeletal and cardiac muscle RyRs provides a structural basis for the regulation of the RyRs by their multiple effectors.

Figure 1.

Open RyR1 channel structure. The structure (PDB code 5TAL) reveals the major domains and the location of Ca²⁺-, ATP-, and caffeine-binding sites.

Figure 2.

Ca²⁺ dependence of RyR1. Data obtained using the planar lipid bilayer and [³H]ryanodine binding methods. With modifications from Heiny and Meissner (2012).

Figure 3.

Ca²⁺ dependence of single purified RyR1, RyR2, and RyR3. Shown are channel P_o values as a function of cytosolic free Ca²⁺ in 250 mM K⁺, pH 7.4 medium. Redrawn from Xu et al. (1998); (RyR1 and RyR2) and Chen et al. (1997); (RyR3).

Figure 4.

Structure of regulatory sites in RyR1. Ca²⁺ (A)-, ATP (B)-, and caffeine (C)-binding sites of open RyR1 (PDB code 5TAL).

Figure 5.

Effect of ryanodine on single purified RyR1. Single-channel recordings of K⁺ current of purified RyR1 incorporated in a planar lipid bilayer. The top trace shows the appearance of an subconductance state with P_o ∼1, several minutes after the addition of 30 µM ryanodine to the cis cytosolic side of the bilayer. The bottom trace illustrates the transition from the subconductance state to a fully closed state within 1 min after the addition of 2 mM ryanodine. Bars on left indicate the open (o) and closed (c) channel. From Lai et al. (1989).