Ca2+ Release Channels Join the 'Resolution Revolution'

Abstract

Ryanodine receptors (RyRs) are calcium release channels expressed in the sarcoendoplasmic reticula of many cell types including cardiac and skeletal muscle cells. In recent years Ca²⁺ leak through RyRs has been implicated as a major contributor to the development of diseases including heart failure, muscle myopathies, Alzheimer's disease, and diabetes, making it an important therapeutic target. Recent mammalian RyR1 cryoelectron microscopy (cryo-EM) structures of multiple functional states have clarified longstanding questions including the architecture of the transmembrane (TM) pore and cytoplasmic domains, the location and architecture of the channel gate, ligand-binding sites, and the gating mechanism. As we advance toward complete models of RyRs this new information enables the determination of domain-domain interfaces and the location and structural effects of disease-causing RyR mutations.

Keywords: calcium channel; cryo-EM; excitation–contraction coupling; ryanodine receptors; structure.

Figure 1. Ca²⁺ handling components in skeletal muscle Excitation-contraction coupling

Upon plasma membrane depolarization DHPR (dihydropyridine receptor) mechanically activates RyR1 to release SR Ca²⁺, which triggers muscle contraction. The Ca²⁺ ATPase SERCA1a pumps Ca²⁺ back into the SR and PMCA (plasma membrane Ca2+ ATPase) pump out the Ca²⁺ from cytoplasm causing muscle relaxation. The RyR1 complex includes: calstabin1, which stabilizes the closed state of the channel, kinases and phosphatases that modulate the channel through phosphorylation (yellow stars marks the phosphorylation site), and calmodulin. RyR1 can be activated by stress pathways via β-adrenergic receptors (β-AR) resulting in increased Ca²⁺ release and enhanced muscle performance. SR Ca²⁺ release may also modulate mitochondrial function, particularly during states of chronic stress (e.g. muscular dystrophy), resulting in generation of reactive oxygen species that can oxidize RyR1 and deplete calstabin1 from the channel, rendering the channels leaky. Leaky RyR1 channels were shown to be a contributing factor to diseases including muscle myopathies, Duchenne muscular dystrophy and cancer.

Figure 2. RyR1 domain assignment

A RyR1 tetramer viewed from the cytosol (left) and from the plane of the membrane. One protomer of the tetramer is shown in colors. B - The RyR1 protomer can be divided into 5 main domains: The NTD is depicted in blue, the three SPRY domains and RYR1&2 in cyan, the Bridging solenoid including Ry3&4 in green, the core solenoid in red and the pore domain in orange. The modulatory subunit calstabin is shown in yellow. A newly identified transmembrane helix TmX is shown in black. The orientation of the 3 SPRY domains in the inset and the location of RY1&2 and RY3&4 are indicated. C – Indicates the sequence boundaries of the 5 main domains.

Figure 3. Clusters of mutation hot spots

Disease-causing RyR1 mutations (http://www.uniprot.org/uniprot/P21817) on the NTD (red spheres), bridging solenoid (blue spheres), core solenoid (purple spheres) and on the transmembrane domain (orange spheres), viewed from the side (left) and from the cytosol (right). Insets show the domain-domain interface between the NTD and the bridging solenoid at the ‘zipping’ point where DP4 (magenta) is located. On the right inset the interaction between NTD-A (cyan) and NTD-B of the adjacent protomer (green) also shows mutations clusters on both sides of this domain-domain interface.

Figure 4. RyR1 gate latch

The CTD forms intimate interactions with the core solenoid TaF domain. Upon Ca²⁺ and ATP binding, the conformational changes in the cytoplasmic shell are coupled to the bowing of the pore S6 helix and the dilation of the pore aperture (Ile4937). Dashed arrows represent the apparent direction of movements from closed to open states. Ca²⁺, ATP and Caffeine, all RyR1 activators, bind at different sites of the CTD-core solenoid inter-domain interfaces. Here one protomer is shown in orange (closed state) and blue (open state).

Figure 5. RyR1 moving parts

Upon Ca²⁺ and ATP binding dilated channel pore aperture can be observed coupled to exo-rotation of the cytoplasmic shell and clockwise translation of the NTD cytoplasmic vestibule. Dashed arrows represent the apparent direction of movements from closed to open states. Similar conformational changes were observed in in the presence of PCB95 or ruthenium red. Curiously, an ~8 Å translation of the EF-hand pair towards the S2–S3 helical bundle is observed also in the RyR1 ‘primed’ state. Insets show enlarged views of the structures at the indicated dashed rectangles.