1.A.3 The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca²⁺ Channel (RIR-CaC) Family

Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP₃)-sensitive Ca²⁺-release channels function in the release of Ca²⁺ from intracellular storage sites in animal cells and thereby regulate various Ca²⁺-dependent physiological processes (Van Petegem 2012). They consist of (1) an N-terminal ligand binding domain, (2) a central modulatory domain and (3) a C-terminal channel-forming domain. The 3-D structure (2.2 Å) of the inositol 1,3,5-triphosphate receptor of an IP₃ receptor has been solved (Bosanac et al., 2002). Structural and functional conservation of key domains in InsP(3) and ryanodine receptors has been reviewed (Seo et al., 2012).  Members of the VIC (1.A.1), RIR-CaC (2.A.3) and TRP-CC (1.A.4) families have similar transmembrane domain structures, but very different cytosolic doman structures (Mio et al. 2008). Ryanodine receptor regulation occurs by intramolecular interaction between cytoplasmic and transmembrane domains (George et al. 2004).

RyR1 activation is regulated by several proteins from both the cytoplasm and lumen of the SR. Chen and Kudryashev 2020 reported the structure of RyR1 (TC# 1.A.3.1.2) from native SR membranes in closed and open states. Compared to previously reported structures of purified RyR1, the new structures reveal helix-like densities traversing the bilayer approximately 5 nm from the RyR1 transmembrane domain and sarcoplasmic extensions linking RyR1 to a putative calsequestrin network. The primary conformation of RyR1 in situ and its structural variations were reported (Chen and Kudryashev 2020). The activation of RyR1 is associated with changes in membrane curvature and movement in the sarcoplasmic extensions. The G4911E mutation in GdRyR may be a potential mechanism for the development of resistance to diamide insecticides in Galeruca daurica (a leaf beetle) (Ren et al. 2022).

Ry receptors occur primarily in muscle cell sarcoplasmic reticular (SR) membranes, and IP₃ receptors occur primarily in brain cell endoplasmic reticular (ER) membranes where they effect release of Ca²⁺ into the cytoplasm upon activation (opening) of the channel. They are redox sensors, possibly providing a partial explanation for how they control cytoplasmic Ca²⁺. Ry receptors have been identified in heart mitochondria, and these provide the main pathway for Ca²⁺ entry (Beutner et al., 2001). Sun et al. (2011) have demonstrated oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca²⁺ release channel (RyR1) by NADPH oxidase 4.

The Ry receptors are activated as a result of the activity of dihydropyridine-sensitive Ca²⁺ channels. Ry receptors, IP₃ receptors, and dihydropyridine-sensitive Ca²⁺ channels (TC#1.A.1.11.2) are members of the voltage-sensitive ion channel (VIC) superfamily (TC#1.A.1). Dihydropyridine-sensitive channels are present in the T-tubular systems of muscle tissues. Ry receptor 2 dysfunction leads to arrhythmias, alterred myocyte contraction during the process of EC (excitation-contraction) coupling, and sudden cardiac death (Thomas et al., 2007). Neomycin is a RyR blocker which serves as a pore plug and a competitive antagonist at a cytoplasmic Ca²⁺ binding site that causes allosteric inhibition (Laver et al., 2007). The cytoplasmic domain of RyR1, which is primarily expressed in skeletal muscle, interacts with Ca²⁺ and Mg²⁺ ions, ligands such as ATP, caffeine and ryanodine, and accessory proteins such as calmodulin (CaM; TC# 8.A.82) (Chen and Kudryashev 2020). CaM in its Ca²⁺-unbound form is a weak agonist of RyR1, while in its Ca²⁺-bound form it is an RyR1 antagonist. A 10-kDa protein, S100A1, capable of increasing the open probability of RyR1, may compete with CaM for the same binding site on the receptor. In the SR lumen, the major Ca²⁺-buffering protein, calsequestrin (CSQ; TC# 8.A.88), interacts with RyR1 indirectly through the membrane-anchored proteins triadin TC# 8.A.28.1.3) and junctin (TC# 8.A.28.1.4), each of which has a single TMS and a disordered intra-SR domain. CSQ has two isoforms: CSQ1, which interacts with RyR1 in skeletal muscle, and CSQ2, which interacts with RyR2, a form primarily expressed in cardiac muscle. CSQ polymerizes in a Ca²⁺-dependent manner and regulates the activity of RyR1. Biochemical analysis suggests that CSQ1 is the major protein component found in the sarcoplasmic reticulum at its junction with T-tubules (Chen and Kudryashev 2020).

Ry receptors are homotetrameric complexes with each subunit exhibiting a molecular size of over 500,000 daltons (about 5,000 amino acyl residues). They possess C-terminal domains with six putative transmembrane α-helical spanners (TMSs). Putative pore-forming sequences occur between the fifth and sixth TMSs as suggested for members of the VIC family. Recently an 8 TMS topology with four hairpin loops has been suggested (Du et al., 2002). The large N-terminal hydrophilic domains and the small C-terminal hydrophilic domains are localized to the cytoplasm. Low resolution 3-dimensional structural data are available. Mammals possess at least three isoforms which probably arose by gene duplication and divergence before divergence of the mammalian species. Homologues are present in Drosophila melanogaster and Caenorabditis elegans. Interactions of RyRs with insecticides and drugs has been reviewed (Sun and Xu 2019).

Tetrameric cardiac and skeletal muscle sarcoplasmic reticular ryanodine receptors (RyR) are large (~2.3 MDa). The complexes include signaling proteins such as 4 FKBP12 molecules, protein kinases, phosphatases, etc. They modulate the activity of and the binding of immunophilin to the channel. FKBP12 is required for normal gating as well as coupled gating between neighboring channels. PKA phosphorylation of RyR dissociates FKBP12 yielding increased Ca²⁺ sensitivity for activation, part of the excitation-contraction (fight or flight) response (Gaburjakova et al., 2001).

RyR1 (TC# 1.A.3.1.2) is an intracellular calcium (Ca²⁺) release channel required for skeletal muscle contraction. des Georges et al. 2016 presented cryo-EM reconstructions of RyR1 in multiple functional states, revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca²⁺, ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca²⁺ alone induces conformational changes in the cytoplasmic assembly ('priming'), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain (des Georges et al. 2016).

IP₃ receptors resemble Ry receptors in many respects (Mikoshiba, 2012). (1) They are homotetrameric complexes with each subunit exhibiting a molecular size of over 300,000 daltons (about 2,700 amino acyl residues). (2) They possess C-terminal channel domains that are homologous to those of the Ry receptors. (3) The channel domains possess six putative TMSs and a putative channel lining region between TMSs 5 and 6. (4) Both the large N-terminal domains and the smaller C-terminal tails face the cytoplasm. (5) They possess covalently linked carbohydrate on extracytoplasmic loops of the channel domains. (6) They have three currently recognized isoforms (types 1, 2, and 3) in mammals which are subject to differential regulation and have different tissue distributions. They co-localize with Orai channels (1.A.52) in pancreatic acinar cells (Lur et al., 2011).

IP₃ receptors possess three domains: N-terminal IP₃-binding domains, central coupling or regulatory domains and C-terminal channel domains. Channels are activated by IP₃ binding, and like the Ry receptors, the activities of the IP₃ receptor channels are regulated by phosphorylation of the regulatory domains, catalyzed by various protein kinases. They predominate in the endoplasmic reticular membranes of various cell types in the brain but have also been found in the plasma membranes of some nerve cells derived from a variety of tissues.

Specific residues in the putative pore helix, selectivity filter and S6 transmembrane helix of the IP3 receptor, have been mutated (Schug et al., 2008) in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel. Channel function was also inactivated by G2586P and F2592D mutations. These studies defined the pore-forming segment in IP (Schug et al., 2008).

The channel domains of the Ry and IP₃ receptors comprise a coherent family that shows apparent structural similarities as well as sequence similarity with proteins of the VIC family (TC #1.A.1). The Ry receptors and the IP₃ receptors cluster separately on the RIR-CaC family tree. They both have homologues in Drosophila. Based on the phylogenetic tree for the family, the family probably evolved in the following sequence: (1) A gene duplication event occurred that gave rise to Ry and IP3 receptors in invertebrates. (2) Vertebrates evolved from invertebrates. (3) The three isoforms of each receptor arose as a result of two distinct gene duplication events. (4) These isoforms were transmitted to mammals before divergence of the mammalian species. 

In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca²⁺ channels and the flux of Ca²⁺ into the cells. This increases Ca²⁺ binding to ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca²⁺, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca²⁺ release events known as 'sparks.' RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Walker et al. 2016 demonstrated that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca²⁺ sparks. They showed that the probability of a Ca²⁺ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s. 

Large-conductance Ca²⁺ release channels, ryanodine receptors (RyRs), mediate the release of Ca²⁺ from the endo/sarcoplasmic reticulum, to the cytoplasm. 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 the brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four approximately 12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases (Meissner 2017). The greatest sequence similarity amoung RyRs is in the C-terminal region that forms the transmembrane, ion-conducting domain of ~500 aas. The remaining approximately 4,500 aas 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 (Meissner 2017).

The generalized transport reaction catalyzed by members of the RIR-CaC family following channel activation is:

Ca²⁺ (out, or sequestered in the ER or SR) → Ca²⁺ (cell cytoplasm).